Autoantibodies and their targets in the diagnosis of peripheral neuropathies

ABSTRACT

The present invention relates to methods of diagnosing peripheral neuropathies that comprise determining the titer of autoantibodies directed toward particular nervous system antigens. It also provides for substantially purified preparations of specific antigens, namely neuroprotein-1, histone H3 (NP-2), β-tubulin (NP-3), neuroprotein-4, neuroprotein-5, NP-9 antigen, and SP neural antigen, which may be used in such diagnostic methods.

This invention was made with government support under grant number AG07438 awarded by the National Institutes of Health. The government hascertain rights in the invention.

This is a Continuation-In-Part Application of U.S. patent applicationSer. No. 08/137,895, filed Oct. 18, 1993, which is aContinuation-In-Part Application of U.S. patent application Ser. No.07/925,926, filed Aug. 7, 1992, now abandoned, which is a ContinuationApplication of U.S. patent application Ser. No. 07/743,005, filed Aug.9, 1991, now abandoned, the entire contents of which are herebyincorporated by reference.

FIELD OF THE INVENTION

The present invention relates to methods of diagnosing peripheralneuropathies that comprise determining the titer of antibodies directedtoward particular nervous system antigens. More specifically, itprovides for substantially purified preparations of a specific antigen,namely neuroprotein-1, histone H3 (neuroprotein-2), β-tubulin(neuroprotein-3), neuroprotein-4, neuroprotein-9 (NP-9), and SP neuralantigen, formerly known as central myelin antigen (CMA), galopin, orGALOP syndrome-related antigen (GRA), which may be used in suchdiagnostic methods.

2. BACKGROUND OF THE INVENTION

2.1. PERIPHERAL NEUROPATHIES

A patient who exhibits a disorder of one or more peripheral nerves issaid to suffer from a peripheral neuropathy. Peripheral nerves extendbeyond the brain and spinal cord into tissues that lie outside thecentral nervous system to provide a bidirectional communication network.They serve as conduits of impulses from the brain and spinal cord to therest of the body; for example, motor neurons carry signals to directmovement. Peripheral nerves are also capable of transmitting sensoryinformation gathered by specialized receptors to the brain. In short,peripheral nerves provide the connection between brain, body, andenvironment, and serve to coordinate the relationship between anorganism's brain and the outside world.

A peripheral neuropathy may manifest itself in a number of ways. If amotor nerve is affected, the patient may exhibit weakness in the musclegroups supplied by that nerve. If a sensory nerve is involved, thepatient may experience numbness, tingling, loss of sensitivity totemperature, touch, and/or vibration, or even increased sensitivity inthe area innervated by the diseased nerve.

Numerous varieties of peripheral neuropathy exist. Some are common,others are extremely rare. The etiology of certain peripheralneuropathies is well understood but some remain a mystery. Manyneuropathies have been classified into particular syndromes. Eachsyndrome is associated with its own set of clinical symptoms and signs,prognosis, and treatment options. It is extremely important to be ableto match a particular patient with the syndrome that corresponds to hisor her clinical condition. Such matching, like a road map, permits thephysician to choose a course of treatment and to counsel the patient asto prognosis. Often the identification of a syndrome alerts thephysician to another medical condition associated with the patient'speripheral neuropathy which requires a particular course of treatmentand carries its own prognosis. Accordingly, the ability to make acorrect and precise diagnosis is exceedingly important in the managementof a patient suffering from a peripheral neuropathy. Making the correctdiagnosis may, however, be difficult. In the past, such diagnosis hasdepended upon an analysis of the patient's symptoms and an extremelydetailed physical examination. To further complicate matters, manyperipheral neuropathy syndromes have not yet been fully characterized.

Peripheral neuropathies may appear as manifestations of a wide varietyof disease processes, including genetic, traumatic, metabolic, immune,and vascular disorders, as shown by Table I (see, for review, Plum andPosner, 1985, in "Pathophysiology The Biological Principles of Disease,"Smith and Thier, eds., Second Edition, W. B. Saunders Co., Philadelphia,Pa., pp. 10851090).

                  TABLE I                                                         ______________________________________                                        ANATOMIC CLASSIFICATION OF PERIPHERAL NEUROPATHY                                TWO OVERALL TYPES                                                                              1. SYMMETRICAL GENERALIZED                                    2. FOCAL AND MULTIFOCAL                                                    ______________________________________                                        1.  Symmetrical Generalized Neuropathies (Polyneuropathies)                           Distal Axonopathies                                                                          Toxic - many drugs, industrial and                        environmental chemicals                                                       Metabolic - uremia, diabetes,                                                 porphyria, endocrine                                                          Deficiency - thiamine, pyridoxine                                             Genetic - HMSN II                                                             Malignancy associated - oat-cell                                              carcinoma, multiple myeloma                                                  Myelinopathies Toxic - diphtheria, buckthorn                                   Immunologic - acute inflammatory                                              polyneuropathy (Guillain-Barre),                                              chronic inflammatory polyneuropathy                                           Genetic - Refsum disease,                                                     metachromatic leukodystrophy                                                 Neuronopathies                                                                somatic motor Undetermined - amyotrophic lateral                               sclerosis                                                                     Genetic - hereditary motor                                                    neuronopathies                                                               somatic sensory Infectious - herpes zoster                                     neuronitis                                                                    Malignancy-associated - sensory                                               neuronopathy syndrome                                                         Toxic - pyridoxine sensory                                                    neuronopathy syndrome                                                         Undetermined - subacute sensory                                               neuronopathy syndrome                                                        autonomic Genetic - hereditary dysautonomia                                    (HSN IV)                                                                   2.  Focal (Mononeuropathy) and Multifocal (Multiple                              Mononeuropathy) Neuropathies                                                  Ischemia - polyarteritis, diabetes, rheumatoid arthritis                      Infiltration - leukemia, lymphoma, granuloma, Schwannoma,                     amyloid                                                                       Physical injuries - serverance, focal crush, compression,                     stretch and traction, entrapment                                              Immunologic - brachial and lumbar plexopathy                               ______________________________________                                    

Neuropathies may be classified on the basis of the anatomic component ofperipheral nerve most affected. For example, some peripheralneuropathies, such as Guillain-Barre syndrome, which is associated withinflammation of peripheral nerve, is classified as a demyelinatingneuropathy because it is associated with destruction of the myelinsheath that normally surrounds the nerve cell axon. In contrast, axonalneuropathies result from damage to the axon caused either by directinjury or, more commonly, from metabolic or toxic injury. In axonalneuropathy, the myelin sheaths disintegrate, as in demyelinatingneuropathy, but myelin loss is secondary to deterioration of the axon.Still other neuropathies, classified as neuronopathies, are caused bydegeneration of the nerve cell body; examples include amyotrophiclateral sclerosis and herpes zoster neuronitis.

Peripheral neuropathies are also classified according to thedistribution of affected nerves. For example, as shown in Table I, someneuropathies are symmetrically, generally distributed, whereas othersare localized to one or several areas of the body (the focal andmultifocal neuropathies).

Yet another characteristic used to categorize peripheral neuropathies isthe nature of the patient's symptoms, i.e., whether the patient sufferspredominantly from sensory or motor abnormalities. Some peripheralneuropathies, such as amyotrophic lateral sclerosis (ALS) and therecently described Multifocal Motor Neuropathy (MMN) with conductionblock are associated primarily with motor dysfunction. Others, such asparaneoplastic sensory neuropathy and neuronopathy associated withSjogren's syndrome, are manifested by sensory abnormalities.

A brief description of several disorders of peripheral nerves asfollows.

2.1.1. AMYOTROPHIC LATERAL SCLEROSIS

Of the predominantly anterior horn cell (AHC) disorders, amyotrophiclateral sclerosis (ALS or Lou Gehrig's disease) is the most common (seeWilliams and Windebank, 1991, Mayo Clin. Proc. 66:54-82 for review).

The initial complaint in most patients with ALS is weakness, morecommonly of the upper limbs (Gubbay et al., 1985, J. Neurol. 232:295-300; Vejjajiva et al., 1967, J. Neurol. Sci. 4:299-314; Li et al.,1988, J. Neurol. Neurosurg. Psychiatry 51:778-784). Usually the earlypattern of weakness, atrophy, and other neurological signs is asymmetricand often focal (Munsat et al., 1988, Neurol. 38:409-413). Musclecramps, paresthesia (tingling sensations) and pain are frequentcomplaints (Williams and Windebank). Widespread fasciculations areusually present (id.). The rate of progression of the disease variesfrom patient to patient (Gubbay et al., 1985, J. Neurol. 232:295-300),but in virtually all cases the disease eventually results in completeincapacity, widespread paralysis (including respiratory paralysis) anddeath.

Anatomically, the most prominent changes are atrophy of the spinal cordand associated ventral roots and firmness of the lateral columns (hencethe name, amyotrophic lateral sclerosis; Williams and Windebank). Uppermotor neurons are also involved and degenerate in ALS. The brain mayappear normal macroscopically, although atrophy of the motor andpremotor cortices is usually present due to upper motor neuroninvolvement. There is widespread loss of Betz cells and other pyramidalcells from the precentral cortex, with consequent reactive gliosis(Hammer et al., 1979, Exp. Neurol. 63:336346).

Current treatment consists of symptomatic therapy to diminish musclecramps, pain, and fatiguability. Prosthetic devices are used tocompensate for muscle weakness. Pharmacologic therapy to alter theprogress of the disease has, however, been largely unsuccessful.Putative therapeutic benefits of thyrotropin releasing hormone have metwith conflicting results (Brooks, 1989, Ann. N.Y. Acad. Sci.553:431-461). Administration of gangliosides has been ineffective(Lacomblez et al., 1989, Neurol. 39:1635-1637). Plasmapheresis has shownno therapeutic advantage, either alone or in combination withimmunosuppressive treatment (Olarte et al., 1980, Ann. Neurol.8:644-645; Kelemen et al., 1983, Arch. Neurol. 40:752-753). Theantiviral agent guanidine was reported to have potential short-termbenefits, but the results were not reproducible (Munsat et al., 1981,Neurol. 31:1054-1055). Administration of branched-chain amino acids toactivate glutamate dehydrogenase was reported to slow the rate ofdecline of patients in an abbreviated study (Plaitakis et al., 1988,Lancet 1:1015-1018). Most recent therapeutic trials, some in progress,involve whole-body total lymphoid irradiation, the use of amino acidsN-acetyl-cysteine, N-acetylmethionine, L-threonine, and long-termintrathecal infusion of thyrotropin releasing hormone (Williams andWindebank).

Animal models that bear clinical and pathologic resemblances to ALSinclude the MND mouse, an autosomal dominant mutant exhibitinglate-onset progressive degeneration of both upper and lower motorneurons (Messer and Flaherty, 1986, J. Neurogen. 3:345-355); the wobblermouse, that exhibits forelimb weakness and atrophy in early life due tomuscle denervation, and hereditary canine spinal muscular atrophy in theBrittany spaniel (Sack et al., 1984, Ann. Neurol. 15:369-373; Silleviset al., 1989, J. Neurol. Sci. 91:231-258; Bird et al., 1971, ActaNeuropathol. 19:39-50).

2.1.2. MULTIFOCAL MOTOR NEUROPATHY WITH CONDUCTION BLOCK

In previous years, patients suffering from multifocal motor neuropathy(MMN) with conduction block were often considered to have pure motorforms of chronic inflammatory demyelinating polyneuropathy (CIDP) orlower motor neuron (LMN) forms of ALS (Bird, 1990, Current OpinionNeurol. Neurosurg. 3:704-707). MMN has recently been characterized as adistinct clinical syndrome. MMN appears to be characterized clinicallyby asymmetric, progressive, predominantly distal limb weakness; arms areinvolved more frequently than legs and there is generally no bulbar,upper motor neuron, or sensory involvement (id.). In more than eightypercent of patients the weakness begins in the hands and may progressslowly for periods up to twenty years. MMN is more common in males thanfemales (2:1) and frequently (66 percent) begins in patients youngerthan 45 years of age. Nerve conduction studies show evidence ofmultifocal conduction block on motor but not on sensory axons (Chad etal., 1986, Neurology 36:1260-1266; Parry and Clarke, 1988 Muscle Nerve11:103-107; Pestronk et al., 1988, Ann. Neurol. 24:73-78).

Patients suffering from MMN appear not to improve clinically withcorticosteroid therapy; Pestronk et al. (1990, Ann. Neurol. 27:316-326)noted improvement in only one out of seven patients treated withhigh-dosage prednisone; treatment with cyclophosphamide appeared to bemore successful. Pestronk et al. (1989, Neurology 39:628-633) havesuggested that prednisone and cyclophosphamide may exert differenteffects on autoantibodies in neuromuscular disorders.

MMN may be distinguishable from another motor neuropathy syndrome thatmore clearly meets criteria for a diagnosis of chronic inflammatorydemyelinating polyneuropathy (CIDP).--Although both are predominantlymotor neuropathies, MMN and motor CIDP differ in their clinicalfeatures, physiologic changes, serologic findings and response toimmunosuppression. In contrast to MMN, patients with motor CIDP usuallyhave symmetric weakness that involves proximal muscles early in thecourse of the disease. While nerve conduction studies in CIDP may showevidence of conduction block, there is often evidence of more diffusedemyelination on both motor and sensory axons. Physiologic changes inmotor CIDP that are found in only a minority of patients with MMNinclude slowing (less than 70% of normal) of conduction velocities, andprolonged distal latencies to the range found in demyelinatingdisorders. High titers of IgM anti-GM1 ganglioside antibodies are onlyrarely found in motor CIDP patients. A further contrast to MMN is theresponse to treatment. As has been reported for the overall populationof CIDP patients, those with motor CIDP often demonstrate increasedstrength within a few weeks to months after treatment with prednisone,plasmapheresis or intravenous human immune globulin.

2.1.3 DISTAL LOWER MOTOR NEURON SYNDROME

Distal Lower Motor Neuron (LMN) Syndrome has a clinical syndrome ofslowly progressive, distal and asymmetrical weakness that begins in aband or foot which is similar to that of MMN (1991 Muscle & Nerve 14:927-936). However, distal LMN begins more frequently in the legs thanMMN and there is an absence of motor conduction block in physiologicstudies of distal LMN. Id.

Patients with distal LMN syndromes, particularly those in the earlystages of the disease or with preserved reflexes in areas of weakness,may be difficult to distinguish from patients with ALS. Id. Distal LMNdiffers clinically from ALS in that distal LMN progresses more slowlythat ALS, patients with distal LMN lack very brisk (4+) reflexes, andthere is a general absence of bulbar dysfunction. Id.

Fifty-five percent of distal LMN patients have high titers of serum IgManti-GM1 ganglioside antibodies, and 15% to 20% of antibody-positivepatients have an associated serum IgM-protein. Id.

Some patients with distal LMN and high anti-GM1 ganglioside antibodytiters improve after treatment with cyclophosphamide or chlorambucil.However, they respond less frequently to immunosuppression than patientswith MMN.

2.1.4. SENSORY NEUROPATHIES

A variety of neuropathies are primarily sensory in nature, includingleprous neuritis, sensory perineuritis, hyperlipidemic neuropathies,certain amyloid polyneuropathies, and distal symmetrical primary sensorydiabetic neuropathy. These are primary axonal or demyelinatingneuropathies.

In addition, pure sensory syndromes, known as sensory neuronopathies,have been identified that result from primary pathological events in thedorsal root ganglion or trigeminal cell bodies (Asbury and Brown, 1990,Current Opinion Neurol. Neurosurg. 3:708-711; Asbury, 1987, Semin.Neurol. 7:58-66). Some examples of sensory syndromes follow.

A severe subacute primary sensory neuropathic disorder may occur in thecontext of concurrent malignancy, particularly small-cell lung cancer,and may in fact precede the diagnosis of malignancy (Asbury and Brown).

Sjogren's syndrome, characterized by dry mucous membranes and skin andthe destruction of salivary and lacrimal glands, appears to beassociated with a sensory neuronopathy. Griffin et al. (1990, Ann.Neurol. 27:304-315) found that eleven women and two men with undiagnosedataxic sensory neuronopathy and autonomic dysfunction all had primarySjogren's syndrome.

Furthermore, hundreds of commonly encountered chemicals, includingenvironmental toxins, vitamins, and various prescription drugs, cancause a polyneuropathy that begins as a distal symmetrical sensoryneuropathy and may progress to a mixed sensory-motor-autonomic disorder.Examples of such chemicals include cis-platinum (Mollman, 1990, N. Engl.J. Med. 322:126-127), vitamin B. (Xu et al., 1989, Neurology39:1077-1083), taxol (Lipton et al., 1989, Neurology 39:368-373) anddoxorubicin (in experimental animals) (Asbury and Brown).

However, the majority of predominantly sensory neuropathies in patientsremain undiagnosed.

2.2. ANTIGENIC STRUCTURES OF THE PERIPHERAL NERVOUS SYSTEM

There is increasing evidence that serum antibodies directed againstglycolipids or glycoproteins (Table II) commonly occur in high titer inpatients with some forms of motor neuron disease and peripheralneuropathy. This association was first noted in patients with chronicdemyelinating neuropathies who had monoclonal IgM serum antibodies thatreacted with myelin-associated glycoprotein. It is now apparent thathigh titers of serum antibodies to GM1 ganglioside commonly occur alongwith lower motor neuron (LMN) diseases and motor neuropathies.Antineuronal antibodies in serum and CSF have been identified inpatients with sensory ganglionopathies and small-cell lung neoplasms. Wewill review the association of clinical neuromuscular syndromes withantibodies that react with glycolipids and structurally relatedglycoproteins.

                  TABLE II                                                        ______________________________________                                        COMMON ANTIGENIC TARGETS                                                        IN NEUROPATHY SYNDROME PATIENTS                                               Compound      Structure                                                     ______________________________________                                        GM1         Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1'Ceramide                              3Neu5Acα2                                         GA1 Galβ2-3GalNAcβ1-4Galβ1-4Glcβ1-1'Ceramide                               .sup. 3 Neu5Acα2                                       GM2 GalNAcβ1-4Galβ1-4Glcβ1-1'Ceramide                                     3Neu5Acα2                                                    Sulfatide SO.sub.4 -3-Galβ1-1'Ceramide                                   MAG and SGPG SO.sub.4 -3-Glucuronic Acid - antigenic epitope                ______________________________________                                         Common antigenic targets in neuropathy syndrome patients. Structures of       GM1 ganglioside, asialoGM1 ganglioside (GA1), and GM2 ganglioside are         illustrated. The gangliosides GM1 and GM2 consist  # of a) a lipid            component, ceramide, b) a carbohydrate moiety (3 sugars for GM2, 4 sugars     for GM1) that includes galactose (Gal), galactosamine (GalNAc) and glucos     (Glc), and c) a sialic acid  # ganglioside. GD1a has an additional sialic     acid attached to the terminal galactose on GM1. GD1b has a second sialic      acid attached to the sialic acid on GM1. GT1B has additional sialic acid      in both locations.                                                       

2.2.1. GANGLIOSIDES

Gangliosides are a family of acidic glycolipids that are composed oflipid and carbohydrate moieties (Table II). The lipid moiety, ceramide,is a fatty acid linked to a long chain base, sphingosine. In mammalianbrain gangliosides, the sphingosine contains 18-20 carbon atoms. Thecarbohydrate portion of gangliosides is a series of 2 or more sugarswith at least one sialic acid. The major gangliosides in mammalian braincontain 1-3 sialic acids, usually N-acetylneuraminic acid contain 1-3sialic acids, usually N-acetylneuraminic acid (Neu5Ac) , and a chain of2-4 other sugars. Four gangliosides are especially abundant in brain,namely GM1, GD1a, GD1b and GT1b. They each contain the same 4 sugarchain (Table II) but vary in the number of sialic acid molecules; GM1ganglioside with one, GD1a and GD1b with two and GT1b with three. Inperipheral nerve a fifth ganglioside, LM1, containing a differentcarbohydrate structure, also occurs in relative abundance. Numerousminor gangliosides in brain, nerve and myelin have been described.Gangliosides generally reside in the outer layer of plasma membrane. Thehydrophilic sugars are located on the outer surface of the membrane.They are linked to the cell by the hydrophobic lipid moiety which isinserted into the membrane.

GM1 ganglioside is one of the most abundant gangliosides in neuronalmembranes but is unusual outside of the nervous system. It has beenpostulated that gangliosides may play a role in membrane and cellfunctions. There is a large amount of literature suggesting thatadministration of exogenous GM1 ganglioside enhances neurite outgrowthand recovery from injury. GM1 ganglioside and other gangliosides canfunction as cellular receptors. The binding of cholera toxin to GM1ganglioside is well documented. Gangliosides on nerve terminals may alsoserve as receptors for tetanus and botulinum toxins.

The abundance of gangliosides in the nervous system and theextracellular location of their sugars suggests that they could beantigenic targets in autoimmune neurological disorders. The terminaldisaccharide on GM1 ganglioside, Galβ-3GalNAc, is known to be antigenicwhen it occurs on systemic glycoproteins. However, the disaccharide onthese glycoproteins is normally hidden from immune attack by a sialicacid attached to each sugar. Several investigators have tested sera frompresumed autoimmune disorders for antibody binding to panels ofgangliosides looking for possible targets of the immune processes.

2.2.2. MYELIN-ASSOCIATED GLYCOPROTEIN

Myelin-associated glycoprotein (MAG; Table II) is a nervoussystem-specific protein that is found in both the central and peripheralnervous systems. It is present in myelin related membranes but not thecompact myelin of oligodendrocytes and Schwann cells. MAG is an integralmembrane protein. Almost one third of its molecular weight is due to thepost-translational addition of carbohydrate molecules. The terminalsulfated glucuronic acid carbohydrate moieties in MAG are importantbecause they are the main targets of IgM paraprotein antibodyreactivity. MAG has structural similarities to immunoglobulins and tocell adhesion molecules. MAG is thought to mediate adhesive and trophicinteractions between cell membranes during myelin formation andmaintenance. Sulfated glucuronic acid epitopes also occur on peripheralnerve glycolipids including sulfated glucuronal paragloboside (SGPG) anda group of glycoproteins of molecular weight 19,000 to 28,000.

2.2.3 HISTONE H3

Histone H3 is a member of a family of basic DNA proteins which arearranged in nucleosomal particles, subcomponents of chromatin. HistoneH3 is found in the inner core of nuclesomes. The protein is composed of134 amino acid residues. An unmodified chain of histone H3 has amolecular weight of 15,117.

2.3. ANTIBODIES IN PERIPHERAL NEUROPATHIES

There has been a growing appreciation that many neurologic disorders mayhave an autoimmune basis. This realization has occurred in conjunctionwith an increasing knowledge of the molecular specificities ofautoantibodies (Steck, 1990, Neurology 40:1489-1492). Consequently, therole of antibody testing as part of the neurologic diagnostic processhas become progressively more important.

2.3.1. ANTI-GM1 GANGLIOSIDE ANTIBODIES

Pestronk et al. (1990, Ann. Neurol. 27:316-326) reports a study of serafrom 74 patients with lower motor neuron syndromes. Antibodyspecificities were compared to clinical and electrophysiological data inthe same patients. Several distinct lower motor neuron syndromes wereidentified based on clinical, physiological, and antiglycolipid antibodycharacteristics. The results indicated that antibodies to gangliosideGM1, to similar glycolipids, and to carbohydrate epitopes on GM1ganglioside and GA1 may be common in sera of patients with lower motorneuron syndromes.

Similarly, Nobile-Orazio et al. (1990, Neurology 40:1747-1750) reports astudy that compared anti-GM1 ganglioside IgM antibody titers byenzyme-linked immunosorbent assay in 56 patients with motor neurondisease, 69 patients with neuropathy, and in 107 control subjects.Anti-GM1 ganglioside IgM antibodies were found in 13 (23 percent) ofmotor neuron disease patients, 13 (18.8 percent) neuropathy patients,and 8 (7 percent) of controls. Two of the 13 neuropathy patientsexhibiting anti-GM1 antibody also were found to have antibodies directedtoward MAG protein.

It appears that high titers of serum IgM anti-GM1 ganglioside antibodies(present at dilutions of >350-400) occur commonly in some motor neuronand peripheral neuropathy syndromes but not in others (Table III). Thehighest titers (>7,000) are especially specific for lower motor neuronsyndromes and multifocal motor neuropathy. Low titers of anti-GM1ganglioside antibodies (<350) are not specific. They may be found insera from patients with a variety of neurologic and autoimmune disordersas well as from some normal controls.

                  TABLE III                                                       ______________________________________                                        IgM ANTI-GM.sub.1 ANTIBODIES - CLINICAL ASSOCIATIONS                          ______________________________________                                        1)    Frequently (>50%) present in high titer (<350):                            Multifocal motor neuropathy                                                   Distal lower motor neuron syndromes                                          2) Occasionally (5-15%) present in high titer:                                 Proximal lower motor neuron syndromes                                         ALS                                                                           Guillain-Barre Syndrome                                                       Polyneuropathies = especially motor-sensory & asymmetric                      Autoimmune disorders without neuropathy                                      3) Rarely (<5%) present in high titer:                                         CIDP                                                                          Sensory neuropathies & neuronopathies                                         Normals (<1%)                                                              ______________________________________                                    

2.3.2. ANTI-MAG ANTIBODIES

High titers of serum antibodies directed against MAG are commonlyassociated with a slowly progressive demyelinating peripheralneuropathy. In 40-50% of patients with IgM monoclonal gammopathy andneuropathy, the M-protein reacts with MAG. The clinical syndrome relatedto high titers of serum anti-MAG antibodies is a distal symmetricneuropathy involving both sensory and motor modalities. Symptoms usuallybegin distally and symmetrically in the feet and legs. The hands arecommonly also affected. Unlike another demyelinating neuropathy, CIDP,weakness only involves proximal musculature late in the disorder.Sensory findings usually include large fiber dysfunction, with sensoryataxia in severe cases. The neuropathy is slowly progressive and mayapparently stabilize for long periods at a point of severe, or onlymild, dysfunction. A majority of patients with IgM anti-MAG relatedpolyneuropathy are male (>80%). Most are older than 50 years of age.Electrophysiological studies usually are indicative of demyelination.The most consistent finding is prolonged distal latencies. Conductionvelocity slowing, temporal dispersion and increased F-response latencyare also seen. Cerebral spinal fluid (CSF) protein concentration isoften elevated. Sera with very high titers of IgM anti-MAG activity showevidence of a monoclonal IgM in many cases, if sensitive screeningmethods, such as immunofixation, are used. In contrast, patients withpredominantly sensory neuropathies, or those that are primarily axonal,only rarely have high-titer serum IgM reactivity to MAG (Nobile-Orazio,et al., 1989, Ann. Neurol. 26:543-550; Dubas et al., 1987, Cas. Rev.Neurol. (Paris) 143:670-683.

A common feature of the anti-MAG antibodies in demyelinating sensorymotor neuropathy syndromes is cross reactivity with compounds that, likeMAG, contain sulfate-3-glucuronate epitopes. These compounds includemyelin components such as the P_(o) glycoprotein (Bollensen et al.,1988, Neurology 38:1266-1270; Hosokawa et al., 1988, InNeuroimmunological Diseases," A. Igata, ed. Tokyo: University of TokyoPress, pp. 55-58) and an acidic glycolipid, sulfate-3-glucuronylparagloboside (SGPG) (Nobile-Orazio; Ilyas et al., 1985, Proc. Natl.Acad. Sci. U.S.A. 82:6697-6700; Chou et al., 1986, J. Biol. Chem.261:11717-11725; Ariga et al., 1987, J. Biol. Chem. 262:848-853).

2.3.3. ANTI-HISTONE H3 ANTIBODIES

Antibodies to Histone H3 have been described in patients with a varietyof autoimmune diseases. In particular, high titers of serum antibodiesdirected against histone H3 are strongly associated with chroniciridocyclitis.

3. SUMMARY OF THE INVENTION

The present invention relates to methods of diagnosing peripheralneuropathies that comprise determining the titer of antibodies directedtoward specific nervous system antigens. It is based on the discoverythat the presence of elevated titers of certain antibodies correlateswith particular clinical and anatomical characteristics.

The present invention, in part, relates to diagnostic methods whichdetermine the presence of antibodies directed toward antigens thatcomprise a SO₄ -3-galactose moiety, including sulfatide antigen. In apreferred embodiment of the invention, the presence of high titers ofanti-sulfatide antibodies in a patient's serum supports a diagnosis of apredominantly sensory axonal neuropathy.

The invention is also based on the discovery and characterization of anumber of nervous system antigens, including neuroprotein-1 (NP-1),histone H3 (neuroprotein-2) (NP-2), β-tubulin (neuroprotein-3) (NP-3),neuroprotein-4 (NP-4), neuroprotein-5 (NP-5), neuroprotein-9 (NP-9), andSP neural antigen, formerly known as central myelin antigen (CMA),galopin, or Galop Syndrome-Related Antigen (GRA). Each of these antigensis recognized by antibodies in patients suffering from peripheralneuropathies, and therefore may be used in diagnostic methods toidentify and define particular neuropathic syndromes.

The invention additionally provides a method of diagnosing particularneuropathies by a triad of high titer antibody activity directed towardGM1 ganglioside and NP-9 antigen, but not toward histone H3(neuroprotein-2).

The correlation between elevated antibody titers toward specificantigens and the clinical and anatomical features of peripheralneuropathies provides an objective standard of diagnosis and allows forthe categorization of patients into groups that share similar prognosesand treatment options. The detection of elevated titers of particularantibodies may serve as an early marker of neurologic disease, and maypermit treatment of the patient's condition before irreversible damagehas occurred. In addition, the characterization of antigen/antibodypairs according to the invention may serve as valuable tools in thestudy of the genesis of peripheral neuropathies.

4. DESCRIPTION OF THE FIGURES

FIG. 1 Western blot of human sera versus myelin proteins (seradilutions=1:1000). Lane 1 illustrates that sera with high ELISA anti-MAGactivity (e.g., patient No. 20) stain MAG on Western blot. Lane 2illustrates that selective antisulfatide sera by ELISA (e.g., patientNo. 3) do not stain MAG. Normal sera at 1:1000 dilution also do notstain MAG.

FIG. 2A Orcinol staining of HPTLC of standards. Lane 1=sulfatidedoublet; Lane 2=bovine brain gangliosides.

FIG. 2B Immunostaining of HPTLC separation of mixture of gangliosidesand sulfatide (sera dilutions=1:1000). Lane 1 illustrates thatantisulfatide sera (e.g., patient No. 3) stain the sulfatide doublet butnot gangliosides. Normal controls and sera that react selectively withMAG using ELISA methodology produce no staining.

FIG. 3 Amino acid sequence of amino terminus of 15-17 kD protein (SEQ IDNO:1) and comparison to histone H3 (SEQ ID NO:2).

FIG. 4 Amino acid sequence (SEQ ID NO:3) of amino terminus ofneuroprotein-3 compared with the amino acid sequence of human β-tubulin(SEQ ID NO:4).

FIG. 5 Western blot of serum (W1160; 1:1000) versus neural proteins.Lanes 2 to 4 illustrate reactivity of serum W1160 with NP-1 bands withmolecular weights of approximately 36 kD, 38 kD, and 42 kD. Molecularweight standards above and below NP-1 are indicated in lane 1.

FIG. 6 Western blot of serum IgM (dilution=1:1000) versus the non-myelinfraction of human CNS (μg protein per lane). Lanes 1 to 5 show bindingof individual anti-GM1 ganglioside sera from patients with treatableMMN. Lanes 6 to 10 show binding of ALS anti-GM1 ganglioside sera. Lanes11 to 15 show binding of peripheral neuropathy, PN, anti-GM1 gangliosidesera. Lane 16 shows binding of a pooled control serum. Note that MMNsera do not bind well to the 17 kD band (histone H3) (arrow). ALS serabind to this band but only unusually to others. PN sera bind to the 17kD band and others with higher or lower molecular weight as well.

FIG. 7 IgM antibody titers versus GM1 ganglioside in patients with MMNresponsive to treatment (MMN Rx), other cases of MMN, LMN syndromes, ALSand peripheral neuropathies (PN). Sera were selected for anti-GM1ganglioside antibody titers >350. Note that there is considerableoverlap between patient groups. The highest titers (>7000) were morecommon in MMN and LMN than in ALS or PN groups.

FIG. 8 IgM antibody titers versus histone H3 (NP-2) in sera with hightiters (>350) of IgM anti-GM1 ganglioside antibodies. Diagnostic groupsare the same as in FIG. 7. Very low titers (<300) were more common inMMN than in the other diagnostic groups. Very high titers (>7000) onlyoccurred in LMN, ALS and PN groups.

FIG. 9 Values of ratios of IGM antibody titers to histone H3 (NP-2)compared to GM1 (histone H3 (NP-2):GM1 ganglioside antibody ratio) inindividual sera. Most MMN sera (82%) have ratios <0.79. Most ALS and PNsera (90%) have ratios >0.79.

FIG. 10 IgM antibodies versus SP neural antigen in 6 patients with Galopsyndrome (O) and 140 controls (o). Dashed line=mean +5 SD of controlserums. Note the very high titer of IgM versus SP neural antigen inGalop syndrome patients.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to autoantibodies and their targets in thediagnosis of peripheral neuropathies. For purposes of clarity, and notby way of limitation, the detailed description of the invention isdivided into the following subsections:

(i) characterization of antigens;

(ii) methods of diagnosis of peripheral neuropathies;

(iii) preparation of antibodies; and

(iv) additional utilities of the invention.

5.1. CHARACTERIZATION OF ANTIGENS

The present invention relates to a number of antigens. In variousembodiments, it provides for antigens that comprise at least one SO₄-3-galactose moiety, including, but not limited to, sulfatide. Inadditional embodiments, the present invention provides for substantiallypurified neuroprotein-1 (NP-1), histone H3 (neuroprotein-2 (NP-2)),β-tubulin (neuroprotein-3 (NP-3)), neuroprotein-4 (NP-4), neuroprotein-5(NP-5), NP-9 antigen, and SP neural antigen.

The present invention provides for substantially purified NP-1, asexemplified in Section 7. Substantially purified NP-1 according to theinvention consists essentially of three related protein molecules havinga molecular weight of about 36, 38 and 42 kD. NP-1 is expressed athigher levels in central nervous system (CNS) and spinal cord non-myelinwhite matter compared to other tissues. It may be prepared, for example,and not by limitation, by processing white matter tissue obtained fromhuman brain by homogenizing white matter in 0.88N sucrose and thencentrifuging the lysate in a discontinuous sucrose gradient with layersof 0.32M and 0.88M sucrose for 30 minutes at 34,000 rpm. The resultingpellet may then be purified by dilipidation in a mixture of ether andethanol at a ratio of about 3:2 for 10 minutes at room temperature andthen washing the pellet three times in 1% Triton-X-100. Afterdilipidation and after the first two washes, each pellet may berecovered by centrifuging at 10,000 rpm for 10-20 minutes. After thethird wash the pellet may be recovered by centrifuging at 20,000 rpm for20 minutes. NP-1 in the pellet may then be taken into solution in 0.1MTris, 0.2 mM PMSF and 0.5 mM EDTA at pH 7.2 and subjected topolyacrylamide gel electrophoresis (PAGE) in a 12 percent polyacrylamidegel to separate its components. Three protein bands, having apparentmolecular weights of about 36, 38 and 42 kD, may then be identified andseparated from the rest of the gel, for example, by cutting out slicesof the gel that correspond to those bands. The NP-1 protein in the bandsmay then be eluted into a suitable buffer using standard techniques.

The present invention further provides for a substantially purifiedneuroprotein-1 having a molecular weight of about 36 kD, a substantiallypurified neuroprotein-1 having a molecular weight of about 38 kD, and asubstantially purified neuroprotein-1 having a molecular weight of about42 kD.

The present invention also provides for substantially purified histoneH3 (NP-2), as exemplified in Section 8. Substantially purified histoneH3 (NP-2) according to the invention consists essentially of two relatedprotein molecules having a molecular weight of about 17-18 kD. HistoneH3 (NP-2) is identifiable as two bands that migrate immediately belowthe large myelin basic protein band on 15% Coomassie blue-stained PAGEof CNS white matter.

Histone H3 (NP-2) is expressed at higher levels in CNS white matter andperipheral nerves compared to other tissues. Histone H3 (NP-2) comprisesthe amino acid sequence substantially as set forth in FIG. 3 (SEQ IDNO:1), or a functionally equivalent sequence. As used herein, the term"functionally equivalent sequence" is construed to mean a sequence inwhich functionally equivalent amino acid residues are substituted forresidues within the sequence resulting in a silent change. For example,one or more amino acid residues within the sequence can be substitutedby another amino acid of a similar polarity which acts as a functionalequivalent, resulting in a silent alternation. Substitutes for an aminoacid within the sequence may be elected from other members of the classto which the amino acid belongs. For example, the nonpolar (hydrophobic)amino acids include alanine, leucine, isoleucine, valine; neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine. The positively charged (basic) amino acidsinclude arginine, lysine and histidine. The negatively charged (acidic)amino acids include aspartic acid and glutamic acid. Based upon itsamino acid sequence, NP-2 appears to be histone H3.

Histone H3 (NP-2) may be prepared, for example, and not by limitation,by processing white matter to produce a nonmyelin pellet as set forthfor NP-1. The pellet may then be purified first by washing in deionizedwater followed by centrifugation at 10,000 rpm, about 8,000 g, for 20minutes and then by dilipidation in a mixture of ether and ethanol at aratio of about 3:2 for 10 minutes at room temperature and then washingin 1% Triton-X-100. After dilipidation and after each wash, the pelletmay be collected by centrifugation at about 10,000 rpm for 10-20minutes. The pellet may then be washed (×3) in Tris buffer pH 7.2containing 0.1 mM PMSF and 0.5 mM EGTA, then collected by centrifugationat 10,000 rpm for 20 minutes. The protein may then be dissolved from thepellet in a solution of 25 mM Chaps, 2M sodium chloride, 1 mM EGTA,0.15M sodium phosphate, 2% glycerol and PMSF by incubation overnight at4° C. Afterward, the dissolved protein may be collected by centrifugingthe product of overnight incubation at 100,000 g (about 34,000 rpm) for2 hours and then recovering the supernatant. The supernatant may then bedesalted and concentrated by micro-ultrafiltration (e.g., AMICON) with a1000 kD filter to form a concentrate that may be subjected to PAGE, forexample, preparative PAGE using a 15% polyacrylamide gel, to separateits components. Two protein bands having apparent molecular weights ofabout 17 to 18 kD may then be identified and separated from the rest ofthe gel, for example, by cutting out slices of the gel that correspondto those bands. The histone H3 (NP-2) protein in the bands may then beeluted into a suitable buffer using standard techniques.

The present invention further provides for substantially purified NP-3,as exemplified in Section 9. Substantially purified NP-3) according tothe invention has a molecular weight of 50-54 kD. It comprises an aminoterminal amino acid sequence substantially as set forth in FIG. 4, (SEQID NO:3), which shows strong homology to β-tubulin (SEQ ID NO:4), and isimmunologically cross-reactive with β-tubulin. On 12% PAGE analysis itmigrates just above the location of Wolfgram proteins in a separation ofhuman white matter or myelin. It may be prepared, for example, and notby limitation, from myelin harvested from human brain according to themethod of Norton and Poduslo, 1973, J. Neurochem, 21: 1171-1191. The CNSmyelin proteins may be purified by dilipidation using a mixture of etherand ethanol at a ratio of 3:2 and then washing first with 1%Triton-X-100 three times. Pellets after each of these washes may beobtained by centrifuging at 10,000 rpm for 10-20 minutes. The protein inthe final pellet so obtained may then be dissolved in 2 percent SDS andthen subjected to PAGE on a 12% polyacrylamide gel. A protein bandhaving an apparent molecular weight of about 50 to 54 kD may then beidentified and separated from the rest of the gel, for example, bycutting out slices of the gel that correspond to those bands. Theβ-tubulin (NP-3) protein in the bands may then be eluted into suitablebuffer using standard techniques.

The present invention also provides for substantially purified NP-4, asexemplified in Section 10. Substantially purified NP-4 has a molecularweight of about 20 to 24 kD. NP-4 may be prepared, for example, and notby limitation by the method as set forth for histone H3 (NP-2), andincluding the washing (×3) in Tris buffer. The pellet is then dissolvedin 2 percent SDS and subjected to PAGE on a 15 percent polyacrylamidegel. A protein band having an apparent molecular weight of about 20-24kD may then be identified and separated from the rest of the gel by themethods outlined for histone H3 (NP-2) and NP-3.

The present invention also provides for substantially purified NP-5, asexemplified in section 11. Substantially purified NP-5 has a molecularweight of about 30-32 kD. It may be prepared for example, and not bylimitation, by differential centrifugation washing and elution ofspecific 30-32 kD bands from PAGE gels using methods similar to thosedescribed for NP-4.

The invention further provides for substantially purified SP neuralantigen, as exemplified in Section 12. SP neural antigen copurifies withmyelin compounds that are soluble in lithium diiodosalicylate. It may beprepared, for example, and not by limitation, by isolation from CNSmyelin by lithium diiodosalicylate (LIS) methodology as previouslydescribed for myelin-associated glycoprotein (MAG) (Quarles et al.,Biochem. J., 1977, 163:635-637; Quarles et al., Biochim. Biophys. Acta,1983, 757:140-143). After this purification, it is then isolated andidentified by thin layer chromatography (TLC). SP neural antigenmigrates as a lipid on TLC plates. Using chloroform: methanol: 0.2%CaCl₂ in water, it migrates in a manner similar to, but not identicalto, total sulfatides. SP neural antigen can be identified on TLC platesby staining with specific human serums from patients with GALOPsyndrome.

The invention further provides for substantially purified histone H3 andNP-9 antigen, as exemplified in Section 12. Substantially purifiedhistone H3 has a molecular weight of 15-17 kD. It may be prepared forexample, and not by limitation, by obtaining a preparation of generalhistones, or, particularly, an arginine rich sub-group (f₃) of histone(for example, Sigma Co. Products, H6005 and H4380, respectively).Histone H3 is separated on a 7.5% PAGE gel, and the appropriate 15-17 kDbands for histone H3 are identified by antibody 5H10 and patient serumW2393 using Western blot. Then, the histone H3 protein in the bands maybe eluted from the PAGE gel using standard techniques, such as thosedescribed for NP-5.

During initial isolation steps, partially purified NP-9 antigencopurifies with myelin glycoproteins, such as myelin-associatedglycoprotein (MAG). Purified NP-9 antigen migrates on thin-layerchromatograms (TLC) as a polar lipid. It may be prepared, for example,and not by limitation, by purification from human brain myelin isolatedfrom the CNS according to the method of Norton and Poduslo, 1973 J.Neurochem. 21: 1171-1191, as set forth for NP-3. The CNS myelin proteinsand NP-9 antigen are purified by serial differential solubility steps.

Lyophilized myelin (1 to 1.2 g) was suspended in chloroform/methanol(2:1, v/v) at a concentration of 10 mg dry wt/ml. Alternatively, thelyophilized myelin can be suspended in hexane:2-propanol (3:2, v/v) at aconcentration of 10 mg dry wt/ml. The suspension was stirred at roomtemperature for about 30 minutes and then centrifuged in teflon (NALGENE3114-0050) tubes with ETFE caps (NALGENE 3131-0024) in a Type 28ultracentrifuge rotor using program #2 (about 30 minutes at 50,000 g,19,000 rpm. Generally, a Sorvall centrifuge was used for rpms of 20,000or less).

The resulting pellet containing NP-9 antigen was washed with diethylether, centrifuged in a Sorvall centrifuge for about 15 minutes at 6,000rpm, then dried under nitrogen. Lithium 3,5-diiodosalicylate (LIS) wasrecrystallized by dissolving 45 g LIS in 100 ml hot water just removedfrom heat after boiling. The solution was mixed thoroughly until nearlyall the LIS was dissolved, then decanted through glass wool to removeparticulate matter. Crystals came out at room temperature in about 4hours. The supernatant was removed by filtration on a Buchner funnelwith a house vacuum, and the crystals were dried under a vacuum to yield60% to 80% recovery.

The residue from the myelin suspension was dispersed in 0.05M Tris/HCl(pH 7.4) containing 0.08M recrystallized LIS with a Dounce homogenizer,using 1 ml Tris/HCl-LIS for each 50 mg dry weight of the lyophilizedmyelin starting material. The suspension was stirred in a cold roomovernight, then about 1.5 volumes of water were added and the mixturewas centrifuged in polycarbonate tubes in a Ti 45 rotor using program #3(about 30 minutes at 78,000 g, 25,0000 rpm) in a ultracentrifuge.

The supernatant provided an enriched source of MAG. The pellet wasdispersed in 0.05M Tris/HCl (pH 7.4) containing 0.3M recrystallized LISwith a Dounce homogenizer using 1 ml Tris/HCl-LIS for each 50 mg dryweight of the lyophilized myelin starting material. The suspension wasstirred in a cold room overnight, then about 1.5 volumes of water wereadded and the mixture was centrifuged in polycarbonate tubes in a Ti 45rotor using program #3 (about 30 minutes at 78,000 g, 25,000 rpm) in anultracentrifuge.

Next, an equal volume of 50% (W/W) phenol was added to the supernatantand stirred at 4° C. for about 45 minutes. The resulting suspension wascentrifuged in corex tubes in a Sorvall centrifuge at 4,000 g, 6,000rpm, for about 45 minutes. Then the mixture was allowed to stand until 2phases formed. The upper phase was dialyzed exhaustively with water toremove the LIS and phenol, then clarified by ultracentrifugation inpolycarbonate tubes in a 45 Ti ultracentrifuge rotor using program #4(100,000 g, 29,000 rpm).

The mixture was then lyophilized, and the lyophilized semipure NP-9antigen was reconstituted using the smallest volume of water possible(1-3 ml). The resulting pellet could be used as semipure NP-9 antigen inELISA assays. Further purification was carried out using thin layerchromatography (TLC) plates (silica gel). Plates were developed inchloroform:methanol:0.2% CaCl₂ in water (55:45:10 by volume).

NP-9 antigen with a maximum concentration present in a spot or band nearand below the position of GM1 ganglioside. NP-9 antigen was specificallyidentified by immunostaining of TLC plates using patient sera #1.0762and GS.

The present invention provides for substantially purified NP-1, histoneH3 (NP-2), β-tubulin (NP-3), NP-4, NP-5, NP-9 antigen, and SP neuralantigen having the characteristics of antigens that are prepared by aprocess exemplified, respectively, in Sections 7, 8, 9, 10, 11, and 12,and briefly described above. However, the present invention alsoprovides for NP-1, histone H3 (NP-2), β-tubulin (NP-3), NP-4, NP-5, NP-9antigen, and SP neural antigen that are prepared by different methods,including different purification strategies, chemical synthesis, andrecombinant DNA technology, etc., provided that the characteristicsexhibited by the respective antigens among Example Sections 7 through 12are substantially retained.

The present invention further provides for fragments and derivatives ofNP-1, histone H3 (NP-2), β-tubulin (NP-3), NP-4, NP-5, NP-9 antigen, andSP neural antigen. Carbohydrate and lipid fragments are also providedfor. Peptide fragments are construed to be at least six amino acids inlength. Derivatives include the products of glycosylation,deglycosylation, phosphorylation, reduction, oxidation, or conjugationof the antigens of the invention to a protein or non-protein molecule.In preferred, nonlimiting embodiments of the invention, the fragment orderivative is immunogenic. In a preferred aspect of the invention, theinvention provides for peptide fragments, carbohydrate fragments, andlipid fragments of SP neural antigen.

The present invention further provides for a substantially purifiedprotein having a molecular weight of about 22 kD that binds tomonoclonal antibody B3H12.

The present invention further provides for a substantially purifiedprotein having a molecular weight of about 10-12 kD that binds to B5G12.

The present invention further provides for:

a substantially purified protein having a molecular weight of about34-38 kD that binds to B4G10;

a substantially purified protein having a molecular weight of about 22kD that binds to B5G12;

a substantially purified protein having a molecular weight of about55-65 kD that binds to B5G10; and

a substantially purified protein having a molecular weight of about15-17 kD that binds to 5H10.

The antibodies of the invention, in particular A1A1.6, A2H3.7, A2H10.1,B3H12, B5G10, B5G12, B5H10, C1F10, C2F3, C1H3, C2H1 and SH10 may be usedto prepare substantially pure preparations of their target antigens byimmunoprecipitation or affinity chromatography.

5.2. METHODS OF DIAGNOSIS OF PERIPHERAL NEUROPATHIES

The present invention provides for methods of diagnosing peripheralneuropathies based upon determining the titer of antibody directedtoward SO₄ -3-galactose, sulfatide, tubulin (preferably, β-tubulin(NP-3)), histone H3 (NP-2), NP-1, NP-4, NP-5, or GM1 ganglioside,histone H3, NP-9 antigen, and SP neural antigen.

According to the invention, a peripheral neuropathy may be diagnosed ina patient by determining that the titer of antibodies in a patientsample directed toward SO₄ -3-galactose, sulfatide, tubulin (preferably,β-tubulin (NP-3)), NP-1, histone H3 (NP-2), NP-4, NP-5, NP-9 antigen, orSP neural antigen is greater than the titer of antibodies which may bepresent in a comparable sample from normal blood, serum, cerebrospinalfluid, nerve tissue, brain tissue, urine, nasal secretions, saliva, orany other body fluid or tissue.

In an alternate embodiment, a peripheral neuropathy may be diagnosed bydetermining that (1) the titers of IgM antibodies in a patient sampledirected toward GM1 ganglioside and NP-9 antigen are greater than thetiter of IgM antibodies which may be present in comparable samples, and(2) the titer of IgM antibodies directed toward histone H3 are less thanthe titer of IgM antibodies which may be present in comparable samples.

Although the following embodiments relate to the determination ofantibody titers in serum, these represent preferred but nonlimitingembodiments of the invention, which may be analogously applied to anypatient sample as described above.

In particular embodiments, the present invention provides for a methodof diagnosing a peripheral neuropathy in a patient comprisingdetermining the titer of antibody that binds to an antigen comprising atleast one SO₄ -3-galactose moiety in a serum sample from the patient, inwhich a high titer correlates positively with a predominantly axonalneuropathy. In preferred embodiments, this neuropathy is predominantlysensory in nature. A high titer of IgG antibody is construed to begreater than about 1:900; if the antibody is IgM, then a high titer isconstrued to be greater than 1:1100. In particularly preferredembodiments of the invention, the antigen comprising SO₄ -3-galactose issulfatide, and the predominantly axonal, predominantly sensoryneuropathy has a clinical history of presenting first as numbness andparesthesia or pain in the feet, and then spreading more proximally inthe legs and eventually involving first the hands and then the arms.Mild weakness may be noted in some patients, but may not begin forseveral months or years after the onset of sensory complaints. onexamination, sensory and motor signs may be more prominent distally.Reflexes may be diminished or absent at the ankles but are usuallypreserved elsewhere.

The present invention further provides for a method of diagnosing aperipheral neuropathy in a patient comprising determining the titer ofantibody that binds to histone H3 (NP-2) in a serum sample from thepatient. In patients with treatable MMN, the ratio of antibody titers tohistone H3 (NP-3) compared to GM1 ganglioside may be less than 0.79. Inpatients with other peripheral neuropathies and ALS this ratio may begreater than 0.79. The difference in ratio may be used to distinguishthe treatable MMN from the essentially untreatable ALS.

The present invention further provides for a method of diagnosing aperipheral neuropathy in a patient comprising determining the titer ofantibody that binds to tubulin (NP-3) in a serum sample from thepatient, in which a titer greater than about 1:1000 correlatespositively with an inflammatory demyelinating polyneuropathy such asGuillain-Barre syndrome or chronic inflammatory demyelinatingpolyneuropathy.

The present invention also provides for a method of diagnosing aperipheral neuropathy in a patient comprising determining the titer ofantibody that binds to neuroprotein-1 in a serum sample from the patientin which a titer greater than or equal to about 1:1000 correlatespositively with a mixed axonal and demyelinating sensorimotorpolyneuropathy (see Section 7).

The present invention also provides for a method of diagnosing aperipheral neuropathy in a patient comprising determining the titer ofantibody that binds to histone H3 NP-2 in a serum sample from a patient,in which a titer greater than 1:1000 and preferably greater than 1:2000combined with the presence of anti-sulfatide antibodies correlatespositively with predominantly sensory or sensory motor signs and axonalor demyelinating neuropathies. Further, the presence of high titers ofantibodies that are cross-reactive with GM1 ganglioside and sulfatidecorrelates positively with a diagnosis of motor neuron disease; "hightiter" in this case should be construed to mean a value of 1:1000 foreither IgM and IgG antibody (See Section 8). In a preferred embodiment,the presence of low titer antibody toward histone H3 (NP-2) and hightiter antibody toward ganglioside GM1, or a ratio of histone H3(NP-2):GM1 ganglioside of less than 0.79 supports a diagnosis of MMN.The presence of high titers of antibody to histone H3 (NP-2) and to GM1ganglioside, or a ratio of titers of histone H3 (NP-2):GM1 gangliosideantibodies of greater than or equal to 0.79 supports a diagnosis of ALSor peripheral neuropathy.

The present invention further provides for a method of diagnosing aperipheral neuropathy in a patient comprising determining the titer ofantibody that binds to β-tubulin (NP-3) in a serum sample from thepatient, in which a titer greater than about 1:1000 correlatespositively with an inflammatory demyelinating polyneuropathy. Inspecific, non-limiting embodiments the inflammatory demyelinatingpolyneuropathy is Guillain-Barre syndrome or chronic inflammatorydemyelinating polyneuropathy (CIDP). As stated in Section 9, antibodiesdirected toward β-tubulin (NP-3) have been observed in high titer at theonset of Guillain-Barre syndrome which decrease over the course of thedisease. Accordingly, the presence of high titers of anti-β-tubulin(NP-3) antibodies may be an early marker of Guillain-Barre Syndrome.High titers of anti-β-tubulin (NP-3) antibodies are present in 40-45percent of patients with CIDP.

The present invention still further provides for a method of diagnosinga peripheral neuropathy in a patient comprising determining the titer ofantibody that binds to NP-4 in a serum sample from the patient in whicha titer greater than or equal to about 1:500 correlates positively witha peripheral neuropathy such as, for example, but not by limitation,Guillain-Barre syndrome or chronic inflammatory demyelinatingpolyneuropathy.

The present invention also provides a method of diagnosing a peripheralneuropathy in a patient comprising determining the titer of IgM antibodythat binds to GM1 ganglioside, the titer of IgM antibody that binds toNP-9 antigen the titer of IgM antibody that binds to histone H3 in aserum sample from the patient. A triad of reactivity comprising thecombination of high serum IgM antibody titers to GM1 ganglioside (>400)and NP-9 antigen (>1600) with relatively low serum IgM antibody titer tohistone H3 (a ratio of H3:GM1 ganglioside of less than <0.79) supports adiagnosis of distal lower motor neuron (D-LMN) syndrome or multifocalmotor neuropathy (MMN).

The present invention also provides a method of diagnosing GALOPsyndrome in a patient comprising determining the titer of antibody thatbinds to SP neural antigen, in which a titer greater than about 1:10,000correlates positively with GALOP syndrome.

According to the present invention, antibody titer may be determined byany method known to the art using standard techniques, including, butnot limited to, enzyme-linked immunosorbent assay (ELISA) and othersolid phase immunoassays, radioimmunoassay, nephelometry, rocketelectrophoresis, immunofluorescence, Western blot (immunoblot), etc. Ina specific, non-limiting embodiment of the invention, antibody titer maybe determined as exemplified in the specific case set forth fordetermining titers to antibodies to glycolipids and MAG in Section6.1.2.

The present invention further provides for diagnostic kits to be usedaccording to the invention. Such kits may comprise (i) substantiallypurified antigen, such as an antigen comprising a SO₄ -3-galactosemoiety, sulfatide, tubulin (NP-3), NP-1, histone H3 (NP-2), NP-4, NP-5,SP neural antigen, or GM1 ganglioside, histone H3 and NP-9 antigen; (ii)detectably labelled antibody "detector antibody" that binds to humanantibody. The detector antibody may comprise an antibody bound to adetectable compound, including, but not limited to, an enzyme,radioactive molecule, or fluorescent compound. In preferred embodimentsof the invention, the detector antibody may be bound to an enzyme thatmay react with an added substrate to yield a colored product; in suchembodiments the kit may preferably include a supply of the substrate. Inan especially preferred embodiment of the invention, the detectorantibody may be conjugated to horseradish peroxidase. Detector antibodymay be specific for a particular class of human antibody, for example,it may bind to human IgM, IgG, IgA, IgE, or IgD, preferably to theconstant region of the molecules. To use the kit, the antigen providedmay be adhered to a solid support and then exposed to serum collectedfrom a patient. The amount of patient antibody bound may then bedetermined using detector antibody. Titers of antibodies may then becalculated from the amount of detector antibody bound using standardconversion algorithms. For example, if detector antibody compriseshorseradish peroxidase, titers of antibody may be calculated as setforth in Pestronk et al. (1990, Ann. Neurol. 27:316-326).

Another object of the present invention is to provide semipurified,purified, and synthetic SP neural antigen.

It is another object of the present invention to provide purifiedmyelin-associated glycoprotein (MAG).

It is yet another object of the present invention to provide a method ofdiagnosing a gait disorder and neuropathy in a patient.

It is still another object of the present invention to provide a methodof diagnosing a predominantly motor syndrome in a patient.

It is another object of the present invention to provide purified andsynthetic NP-9 antigen.

It is yet another object of the present invention to provide a kit fordiagnosing a neuropathy, with or without a gait disorder.

It is still another object of the present invention to provide a methodfor producing an animal model system for neuropathy.

It is another object of the present invention to provide an antibodywhich specifically binds to SP neural antigen.

It is yet another object of the present invention to provide an antibodywhich specifically binds to histone H3.

5.3. PREPARATION OF ANTIBODIES

According to the invention, SO₄ -3-galactose containing antigen,sulfatide, tubulin (preferably β-tubulin (NP-3)), NP-1, histone H3(NP-2), NP-4, NP-5, NP-9 antigen, or SP neural antigen, or fragments orderivatives thereof, may be used as immunogens to generate antibodies.

To improve the likelihood of producing an immune response, the aminoacid sequence of a neuroprotein antigen or the carbohydrate or lipidmoieties may be analyzed in order to identify portions of the moleculewhich may be associated with increased immunogenicity. For example, theamino acid sequence may be subjected to computer analysis to identifysurface epitopes. Alternatively, the deduced amino acid sequences of aneuroprotein antigen from different species could be compared, andrelatively non-homologous regions identified; these non-homologousregions would be more likely to be immunogenic across various species.

For preparation of monoclonal antibodies directed toward the antigens ofthe invention, any technique which provides for the production ofantibody molecules by continuous cell lines in culture may be used. Forexample, the hybridoma technique originally developed by Kohler andMilstein (1975, Nature 256:495-497), as well as the trioma technique,the human B-cell hybridoma technique (Kozbor et al., 1983, ImmunologyToday 4:72), and the EBV-hybridoma technique to produce human monoclonalantibodies (Cole et al., 1985, in "Monoclonal Antibodies and CancerTherapy," Alan R. Liss, Inc., pp. 77-96) and the like are within thescope of the present invention.

The monoclonal antibodies may be human monoclonal antibodies or chimerichuman-mouse (or other species) monoclonal antibodies. Human monoclonalantibodies may be made by any of numerous techniques known in the art(for example, Teng et al., 1983, Proc. Natl. Acad. Sci. U.S.A.80:7308-7312; Kozbor et al., 1983, Immunology Today 4:72-79; Olsson etal., 1982, Meth. Enzymol. 92:3-16). Chimeric antibody molecules may beprepared containing a mouse (or other species) antigen-binding domainwith human constant regions (Morrison et al., 1984, Proc. Natl. Acad.Sci. U.S.A. 81:6851, Takeda et al., 1985, Nature 314:452).

Various procedures known in the art may be used for the production ofpolyclonal antibodies to epitopes of the antigens of the invention. Forthe production of antibody, various host animals can be immunized byinjection with antigen, or fragment or derivative thereof, including butnot limited to rabbits, mice, rats, etc. Various adjuvants may be usedto increase the immunological response, depending on the host species,and including but not limited to Freund's (complete and incomplete),mineral gels such as aluminum hydroxide, surface active substances suchas lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (Bacille Calmette-Guerin) and, CorynebacteriumParvum.

Antibody molecules may be purified by known techniques, for example,immunoabsorption or immunoaffinity chromatography, chromatographicmethods such as HPLC (high performance liquid chromatography), or acombination thereof, etc.

Antibody fragments which contain the idiotype of the molecule can begenerated by known techniques. For example, such fragments include butare not limited to: the F(ab)'₂ fragment which can be produced by pepsindigestion of the antibody molecule; the Fab' fragments which can begenerated by reducing the disulfide bridges of the F(ab)'₂ fragment, andthe 2 Fab or Fab fragments which can be generated by treating theantibody molecule with papain and a reducing agent.

As exemplified in Sections 14, 15, 16, and 17, a number of monoclonalantibodies directed toward antigens of the invention have been produced,including monoclonal antibodies A1A1.6, A2H3.7, and A2H10.1, to NP-1;B3H12, B5G10, and B5G12, and NP-2; C1F10, C2F3, C1H3, and C2H1 to NP-3;and 5H10 to histone H3. The present invention provides for theseantibodies and hybridomas that produce these antibodies and theirfunctional equivalents. Functional equivalents of a monoclonal antibodyare construed herein to refer to antibodies or antibody fragments thatcompetitively inhibit the binding of a monoclonal antibody to its targetantigen. The present invention also provides for fragments andderivatives of the antibodies of the invention.

Antibody produced by these methods may be used to bind to the antigensof the invention in vitro or in vivo. The use of such antibodies mayreveal aberrancies in the distribution or level of expression of theantigens of the invention; for example, peripheral nerve may be found tobe depleted of a particular antigen or may exhibit an overabundance ofan antigen in various peripheral neuropathies. Accordingly, theantibodies of the invention may be used in the diagnosis of peripheralneuropathies. For example, such antibodies may be applied to a samplewhich is a section of peripheral nerve or other tissue or fluid obtainedfrom a patient; if the level of antibody binding to antigen in thesample from the patient differs from the level of binding to acomparable sample from a normal, healthy person, the patient may sufferfrom a peripheral neuropathy or related condition.

The antibodies of the invention may also be used as antigens themselvesto produce anti-idiotype antibody that may be useful in the treatment ofcertain peripheral neuropathies.

The antibodies of the invention may be administered to a non-humananimal in order to produce a model system that may be used to study aperipheral neuropathy.

5.4. ADDITIONAL UTILITIES OF THE INVENTION

In additional embodiments, the present invention may be used to createnon-human animal model systems for peripheral neuropathy and may be usedtoward the cloning and recombinant expression of the neuroproteinantigens of the invention.

In order to create non-human animal model systems for peripheralneuropathy, an antigen of the invention, such as an antigen thatcomprises at least one SO₄ -3-galactose moiety, sulfatide, histone H3(NP-2), tubulin (NP-3), NP-1, NP-4, NP-5, GM1 ganglioside, NP-9 antigen,or SP neural antigen may be used to immunize a non-human animal usingstandard techniques. It may be useful to administer the antigen inconjunction with an immune adjuvant, as set forth in section 5.3. Incases where a peripheral neuropathy is caused or exacerbated by antibodydirected toward an antigen of the invention, animals that produceantibodies against these antigens may produce a peripheral neuropathycomparable to the human condition. In a preferred embodiment of theinvention exemplified in Section 12, a non-human animal immunized withsulfatide mixed with either methylated bovine serum albumin or keyholelimpet hemocyanin (KLH) in complete Freund's adjuvant may serve as amodel system for a peripheral neuropathy associated with axonaldegeneration and weakness.

Further, the antigens of the invention, namely NP-1, histone H3 (NP-2),β-tubulin (NP-3), NP-4, NP-5, NP-9 antigen, and SP neural antigen may becloned and characterized using standard molecular biology techniques.For example, a portion of a protein may be sequenced, (e.g. sequence ofβ-tubulin (NP-3) as depicted in FIG. 3) (SEQ ID NO:1) and that aminoacid sequence may be used to deduce degenerate oligonucleotide probesthat may be used directly to screen genomic or, preferably, cDNAlibraries for a clone that contains protein-encoding sequences, or maybe used in polymerase chain reaction to amplify protein-encodingsequences for subsequent cloning. Once a protein or antigen encodingsequence has been cloned, it may be engineered into an appropriateexpression vector so as to enable the production of recombinant NP-1,histone H3 (NP-2), β-tubulin (NP-3), NP-4, NP-5, or NP-9 antigen inquantity. Such recombinant protein may be used, for example, in thediagnostic methods of the invention.

6. EXAMPLE: POLYNEUROPATHY SYNDROME ASSOCIATED WITH SERUM ANTIBODIES TOSULFATIDE AND MYELIN-ASSOCIATED GLYCOPROTEIN

6.1. MATERIALS AND METHODS

6.1.1. PATIENTS

We tested for antibodies to compounds with sulfated carbohydrate(S-carb) moieties in sera from 64 patients in our neuromuscular clinicpopulation who had acquired neuropathies with prominent sensoryinvolvement. Sera from 35 normals and blood bank volunteers, from 21patients with chronic inflammatory demyelinating polyneuropathies (CIDP)with mainly motor involvement and from 20 patients with amyotrophiclateral sclerosis (ALS) were used to establish a range of normal controland disease control values. For each of the 64 sensory neuropathypatients we determined the pattern and degree of sensory and motor loss(Table IV). We also examined electrophysiologic data obtained as part oftheir clinical evaluation. These studies were characterized according toconventional criteria (Nobile-Orazio et al., 1989, Ann. Neurol.26:543-550; Kelly, 1983, Muscle Nerve 6:504-509) as indicative ofpredominantly axonal degeneration, or demyelination, or a mixture ofboth.

6.1.2. ELISA ANTIBODY ASSAYS

Serum was assayed for antibodies to glycolipids and MAG using ELISAmethodology. Glycolipid antigens and chondroitin sulfates were obtainedfrom Sigma (St. Louis, Mo.). Purified MAG (Quarles, 1988, in "Neuronaland Glial Proteins: Structure, Function and Clinical Applications",Marangos, Campbell and Cohen, eds., Academic Press, Petaluma, Calif.,pp. 295-320; Quarles et al., 1983, Biochem. Biophys. Acta. 757:140-143)was a gift from Dr. Richard H. Quarles (NIH). Purified P_(o) protein wasa gift from Dr. Gihan Tennekoon. Substrates were attached to wells ofmicroliter plates by two methods (Pestronk et al., 1990, Ann. Neurol.27:316-326). For glycolipids 400 ng in 50 μl of methanol was added towells and evaporated to dryness. Approximately 50 ng MAG orapproximately 200 ng of P_(o) protein and chondroitin sulfate in 100 μlof 0.01M phosphate buffered saline (PBS) pH 7.2 with 0.15M NaCl wereadded to wells and incubated overnight at 4° C. Any remaining bindingsites were blocked with 100 μl of 1% human serum albumin in PBSovernight at 4° C. Plates of MAG but not of glycolipids were then washed5 times with 1% bovine serum albumin (BSA) and 0.05% Tween-20 in PBS.

Subsequent steps were performed at 4° C. Between steps, washing (×5) wasperformed using PBS with 1% BSA without detergent. All sera were testedin duplicate. Serum was examined by adding 100 μl of dilutions(1:100-1:200,000 in PBS with 1% BSA) to wells for 5 hours (overnight forMAG). The binding of immunoglobulin to glycolipids or MAG was measuredusing overnight (2 hours for MAG) exposure to specific goat anti-humanIgM or IgG linked to horseradish peroxidase (Cappell-Durham, N.C.) inPBS with 1% BSA (working dilution 1:20,000). Color was developed byadding 100 μl substrate buffer (0.1M citrate buffer pH 4.5 with 0.004%H₂ O₂ and 0.1% phenylenediamine) for 20-50 minutes until a standardpositive control at a 1:1000 dilution reached an optical density (OD) of0.6 above that of normal controls. OD was then determined for the testand control sera at 450 nm. The average OD of normal control sera wassubtracted from the average OD of test sera at each dilution. Titers ofantibodies were calculated from OD data as described in Pestronk et al.(1990, Ann. Neurol. 27:316-326). Readings in the linear range of OD data(0.040 to 0.220 above control) were extrapolated to the value that mightbe expected at a standard dilution of 1:100, multiplied by 1,000 andaveraged. For example, in the test for IgM versus sulfatide in serumfrom patent 6, dilutions of 1:3000 and 1:9000 gave OD readings of 0.150and 0.056 respectively. Using our formula, ##EQU1## we calculated atiter of IgM versus sulfatide of 4,770. In general, a serum with a hightiter of x was detectable (>3 standard deviations (SD) over negativecontrols) in our assays up to a dilution of at least 1/x. We designatedhiqh titers as those greater than 3 SD above the mean value in our 35patient normal control panel. Our results showed that values ≧900 unitswere high for IgG antibodies against sulfatide and MAG and values ≧1100were high for IgM antibodies against sulfatide and MAG.

6.1.3. IMMUNOBLOT ASSAYS

Central nervous system myelin was prepared from human brain (Norton andPoduslo, 1973, J. Neurochem. 21:1171-1191). Myelin proteins (100 μg perlane) were fractionated using 12% SDS polyacrylamide gel electrophoresisand transferred onto nitrocellulose sheets (Towbin et al., 1979, Proc.Natl. Acad. Sci. U.S.A. 74:4350-4354). Test sera were diluted1:1000-1:4000 in PBS with 1% BSA and then incubated with nitrocellulosestrips overnight at 4° C. After washing ×5 using PBS with 1% BSA, thebinding of immunoglobulin was measured using 2-3 hour exposure to goatanti-human IgM linked to horseradish peroxidase in PBS with 1% BSA(working dilution=1:1000). Color was developed with 0.05%diaminobenzidine (DAB) and 0.01% H₂ O₂ in PBS.

6.1.4. IMMUNOSTAINING AFTER HIGH-PERFORMANCE THIN-LAYER CHROMATOGRAPHY(HPTLC)

Sulfatides mixed with a preparation of bovine brain gangliosides (Sigma)were separated by HPTLC on aluminum-backed silica gel 60 HPTLC plates(Merck, Darmstadt, West Germany) using a chloroform: methanol: water(70:30:4) solvent. IgM reactivity in patient sera (1:1000) was detectedby incubation with sera at 4° C. overnight and staining with peroxidaselinked second antibodies and DAB as above.

6.2. RESULTS

6.2.1. SERUM ANTIBODY TESTING

We performed ELISA testing for antibodies to sulfatide and MAG in seraof 64 patients with peripheral neuropathy syndromes characterized byprominent sensory involvement. Table IV summarizes the findings in 22patients with high titers of antibodies to at least one of the twoantigens. Eighteen patients had high titers of serum antibodies thatreacted with sulfatide. In twelve patients, the high titers ofanti-sulfatide antibodies were IgM and in six patients they were IgG. Inthree of the five patients with the highest titers, an IgM paraproteinwas detectable in serum by immunofixation electrophoresis.

Sixteen patients had high titers of anti-MAG antibodies. Thirteen ofthese were IgM class and three were IgG class antibodies. Five of thesix patients with the highest titers had IgM paraproteins.

There appeared to be no correlation between ELISA titers ofantisulfatide and of anti-MAG antibody reactivity in individualpatients. Seven sera (samples 1-7) demonstrated high titer antibodyreactivity only to sulfatide, and four sera (samples 19-22) reacted onlyto MAG. Even the highest titer antibodies to MAG or sulfatide often hadno high titer reactivity to the other antigen. Although there was nocorrelation between titers, half of the sera (11/22) with high levels ofantibody reactivity to one antigen also had high levels to the other.However, three of these sera had IgM reactivity to one antigen but onlyIgG reactivity to the other.

The differential reactivity of the sera that had high ELISA titers onlyto sulfatide or only to MAG was also apparent using overlay methods. Wetested all eleven of these sera (patients 1-7 and 19-22) by Western blotand HPTLC. FIG. 1 shows a comparison of Western blot reactivity ofsample sera with different high titer antibodies as measured by ELISA.The sera with high IgM anti-MAG activity (samples 19-22) stronglystained a protein band (in a CNS myelin protein preparation) thatcorresponds to the molecular weight of MAG (Quarles, 1988, in "Neuronaland Glial Proteins: Structure, Function and Clinical Applications",Marangos, Campbell and Cohen eds., Academic Press, Petaluma, Calif., pp.295-320; Quarles et al., 1983, Biochim. Biophys. Acta, 757:140-143).Sera (samples 1-7) with high ELISA anti-sulfatide activity but no ELISAanti-MAG activity did not stain the MAG band. on HPTLC, high titeranti-sulfatide sera (samples 1-7) stained a doublet band correspondingto sulfatide but not the other glycolipids on the plate (FIG. 2).Selective anti-MAG sera (samples 19-22) stained the sulfatide bandweakly or not at all.

We tested several sera in order to determine whether there was arelationship between titers of antibodies to sulfatide and to otherneuropathy-related antigens (Table V). Several antigens were testedincluding: chondroitin sulfate C, a glycosaminoglycan that has beenassociated with axonal sensory-motor neuropathies (Sherman et al., 1983,Neurology 33:192-201; Yee et al., 1989, Acta Neuropathol. 78:57-64);chondroitin sulfate A, another glycosaminoglycan; P_(o) protein, aperipheral myelin glycoprotein that may react with anti-MAG antibodies(Bollensen et al., 1988, Neurology 38:1266-1270) and GM1 ganglioside andasialo-GM1 (GA1), glycolipids that may be associated with motorneuropathies (Nobile-Orazio et al., 1989, Ann. Neurol. 26:543-550;Pestronk et al., 1990, Ann. Neurol. 27:316-326; Latov, 1987, in"Polyneuropathies Associated With Plasma Cell Dyscrasia", Kelly, Kyleand Latov, eds., Martinus Nijhoff, Boston, Mass., pp. 51-72; Freddo etal., 1986, Neurology 36:454-458; Steck et al., 1987, Ann. Neurol.22:764-767). We found that there was no correlation betweenanti-sulfatide titers and reactivity to chondroitin sulfate A or C,P_(o) protein, GM1 ganglioside or asialo-GM1.

                  TABLE IV                                                        ______________________________________                                               Disease                   Antibody                                        Dura-   titers                                                                tion Clinical Nerve vs.                                                      Age/Sex (yrs) Syndrome Physiology Sulfatide MAG                             ______________________________________                                         1) 38F                                                                              12      S-Pan    Ax(53;NR)                                                                              1,230  --                                          (IgG)                                                                      2) 68M 1 S-Pan; AX(46;38) 1,720 --                                             mild M                                                                       3) 47M 1 S-Pan N(57;58) 232,350*  --                                          4) 29F 2 S-Pan; M(33;NR) 1,230 --                                              mod M  (IgG)                                                                 5) 44F 5 S-Pan; Ax(56;NR(U))   902 --                                          mod M                                                                        6) 69M 1 S-Pan M(41(P);30)  4,770* --                                         7) 60M 5 S-Pan; M(50;30) 1,365 --                                              mild M  (IgG)                                                                8) 59M 1 S-Pan; Ax(45(P);43) 2,547 1,232                                          (IgG)                                                                     9) 52F 1 S-Pan; Ax(40(P);NR) 7,520 1,936                                     10) 68M 4 S-Pan; D(31;NR) 1,920 2,108                                           Mod M                                                                       11) 54M 3 S-Pan; M(37(P);39) 1,392 2,000                                        Mod M                                                                       12) 33M 4 S-Pan; M(42;NR) 3,872 3,776                                           sev M                                                                       13) 75F 1 S-SF; D(14;NR) 14,720  1,128                                          mild M                                                                      14) 44F 1 S-Pan; M(41;NR) 2,136 2,360                                           mild M                                                                      15) 66F 2 S-Pan; M(37;NR) 1,904 174,000*                                        mod M  (IgG)                                                                16) 72M 5 S-Pan; D(17(U);NR) 1,206 200,000*                                     mild M  (IgG)                                                               17) 59M 5 S-Pan; D(16(U);NR)  7,848*  4,416*                                    mild M                                                                      18) 53F 15 S-Pan; D(43;34) 1,054 2,048                                          mild M                                                                      19) 55F 9 S-Pan M(41;MR) -- 22,000*                                           20) 61M 5 S-Pan; D(35;NR) -- 8,480                                              mild M                                                                      21) 69M 10 S-Pan; D(29;NR) -- 205,056*                                          mod M                                                                       22) 63M 4 S-Pan Ax(54;52) -- 1,096                                                 (IgG)                                                                  ______________________________________                                         Sensory and sensorymotor syndrome patients with high titers of antibodies     to sulfatide or MAG. Age is at the time of serum testing.                     Clinical syndrome: S = sensory; Pan = large and small sensory fiber           modalities involved on examination; SF small fiber sensory  # modalities      involved; M = motor; sev = severe weakness (3 out of 5 or less) in at         least one muscle group; mod. = moderate weakness  # (4 out of 5 or worse)     in at least one muscle group; mild = weakness but not worse than 4+ out o     5.                                                                            Nerve Physiology: Ax = axonal; M = mixed, moderate features of axon loss      and demyelination; D = demyelination (Kelly, 1983, Muscle  # Nerve 6:         504-509); N = Normal. Numbers in parenthesis: (A;B)  A = motor conduction     velocity; B = sensory conduction velosity.  # Unless otherwise noted moto     conductions are from median nerve, sensory values from sural. U = ulnar,      = common peroneal, N.R. = no response.                                        Antibody titers: sera with a high titer of x units were generally             significantly above background at a dilution of X. We have listed all         values considered  # high (see methods) for IgM and IgG against sulfatide     and MAG. * = monoclonal IgM paraprotein detected by immunofixation.           Antibodies were IgM unless  # noted. -- = no high titer antibodies            detected.                                                                

6.2.2. CORRELATIONS BETWEEN ANTIBODY REACTIVITY AND CLINICAL ANDPHYSIOLOGICAL PATTERNS

Eleven of thirteen patients with high ELISA titers of IgM anti-MAGantibodies (samples 9-21) had a combined sensory plus motor neuropathy(Table IV). Sensory loss usually involved both large and small fibermodalities. Motor findings were often mild but were unequivocallypresent in eleven patients in this group. The distribution ofsensory-motor loss was always greater distally than proximally. Mostoften the signs were symmetric. However, three patients showedconsiderable asymmetry in strength. Nerve conduction studies revealedsome demyelinating features in twelve of the thirteen patients with highIgM anti-MAG antibodies. Seven had predominantly demyelinating changes.Five had mixed demyelinating and axonal abnormalities. Two patients(samples 7 and 8) had high titers of IgG, but not IgM, anti-MAGantibodies. Both had axonal, sensory polyneuropathies.

In the group of eight patients with high ELISA titers of IgM or IgGanti-sulfatide antibodies but without high titer IgM anti-MAG reactivity(samples 1-8) there were four pure sensory and four sensory plus motorpolyneuropathies. In all these patients sensory loss was distal andinvolved both large and small fiber modalities. Nerve conduction studiesshowed only axonal abnormalities in four patients, mixed features inthree and were normal in one. No patient with selective anti-sulfatideactivity had predominantly demyelinating changes.

None of the sera from 35 normal controls had titers of IgG to MAG orsulfatide ≧900, or titers of IgM to MAG or sulfatide ≧1100.

None of the twelve patients with dorsal root ganglioneuropathy syndromeshad high titers of antibodies to sulfatide or MAG. In other neurologicdisease control groups, none of the 20 patients with ALS or the 21 withmotor CIDP had high titers of antibodies to sulfatide or MAG.

6.3. DISCUSSION

6.3.1. PATIENTS WITH ANTI-SULFATIDE ANTIBODIES

Our eight patients with high titer serum reactivity to sulfatide,without high titer IgM binding to MAG, had similar clinical syndromes ofpredominantly sensory neuropathy (Table IV). At onset these patientsnoted numbness and paraesthesia or pain in the feet. Symptoms usuallyspread more proximally in the legs and appeared in the hands within ayear of onset. Mild weakness was noted in some patients, but usuallybegan several months to years after the onset of sensory complaints. Onexamination sensory and motor signs were more prominent distally.Reflexes were diminished or absent at the ankles but usually preservedelsewhere. Nerve conduction studies generally showed changes compatiblewith axonal disease but only minor, if any, evidence of demyelination.The incidence of high titers of anti-sulfatide antibodies in a generalpopulation of patients with similar idiopathic axonal sensorimotorneuropathies appears to be at least about 20-30 percent.

6.3.2. PATIENTS WITH IgM ANTI-MAG ANTIBODIES

Sensory symptoms and signs were also a common feature in the anti-MAGneuropathy group (Table IV). However, the patients with high titers ofIgM anti-MAG antibodies differed from the anti-sulfatide group in tworespects.

First, mild to moderate weakness was more common in these patients.Distal weakness was present in 85% (11 of 13) of our patients with highIgM anti-MAG titers. Weakness has also been reported in most previouslydescribed patients with IgM anti-MAG antibodies (Nobile-orazio et al.,1989, Ann, Neurol. 26:543-550; Steck et al., 1987, Ann. Neurol.22:764-767; Jauberteau et al., 1988, Rev. Neurol. (Paris) 144:474-480;Kelly et al., 1988, Arch. Neurol. 45:1355-1359; Vital et al., 1989, ActaNeuropathol. 79:160-167; Hafler et al., 1986, Neurology 36:75-78).However, only 44% (four out of nine) of the other antibody-positivepatients in our series had weakness.

Second, patients with high titers of IgM anti-MAG antibodies frequentlyhad some physiologic evidence of demyelination (92%; 12 of 13; Table IV)(Nobile-Orazio et al., 1989, Ann, Neurol. 26:543-550; Steck et al.,1987, Ann. Neurol. 22:764-767; Jauberteau et al., 1988, Rev. Neurol.(Paris) 144:474-480; Kelly et al., 1988, Arch. Neurol. 45:1355-1359;Vital et al., 1989, Acta Neuropathol. 79:160-167; Hafler et al., 1986,Neurology 36:75-78). A majority (54%; 7 of 13) showed predominantlydemyelinating changes (Kelly, 1983, Muscle Nerve 6:504-509). Incontrast, the patients with only anti-sulfatide antibodies hadpredominantly axonal changes; there was some physiologic evidence ofdemyelination in only 43% (3 of 7) and none had a pattern of predominantdemyelination.

6.3.3. PATIENTS WITH ANTI-S-CARB ANTIBODIES

The results of this study provide evidence that antibodies directedagainst compounds containing S-carb moieties are a frequent feature ofperipheral neuropathies with a prominent sensory component. Thissuggests that a compound containing S-carb may be an antigenic markerthat is particularly abundant on axons or myelin of peripheral sensorynerves. However, the fine specificity of anti-S-carb antibodies seems tovary according to the clinical syndrome. In demyelinating sensory-motorneuropathies, the anti-S-carb antibodies tend to cross react withcompounds containing an SO₄ -3-glucuronic acid as the terminal sugar onthe carbohydrate moiety (Nobile-Orazio et al., 1989, 26:543-550; Latov,1987, in "Polyneuropathies Associated With Plasma Cell Dyscrasia",Kelly, Kyle, Latov, eds., Boston, Martinus Nijhoff, pp. 51-72; Steck etal., 1987, 22:764-767; Bollensen et al., 1988, Neurology 38:1266-1270;Hosokawa et al., 1988, in "Neuroimmunological Diseases", A. Igata ed.,Tokyo: University of Tokyo Press, pp. 55-58; Ilyas et al., 1985, Proc.Natl. Acad. Sci. U.S.A. 82:6697-6700). In the patients described herewith predominantly axonal sensory polyneuropathies, anti-sulfatideantibodies may be directed against an epitope that includes an SO₄-3-galactose moiety. Although these sulfated epitopes appear similar,antibodies to one commonly do not cross-react well with the other (TableV; Jauberteau et al., 1989, Neuroscience Letters 97:181-184). This wastrue for six of our seven sera with monoclonal proteins and 11 of 22sera overall. The specificity of both types of anti-S-carb antibodies isfurther shown by their lack of general reactivity with other CNSglycolipids or glycoproteins as measured by ELISA, HPTLC andimmunoblotting studies (FIGS. 1, 2A and 2B; Nobile-Orazio et al., 1989,Ann. Neurol. 26:543-550; Jauberteau et al., 1989, Neuroscience Letters97:181-184).

Others have described patients with anti-MAG antibodies who did not haveserum paraproteins (Nobile-orazio et al., 1989, Ann. Neurol. 26:543-550;Nobile-Orazio et al., 1984, Neurology 34:218-221); however, reports ofsuch patients are rare. In contrast, only 7 of 22 patients in our serieswith high titers of anti-S-carb antibodies had detectable paraproteins.Thus, the frequency of high titer anti-MAG and anti-sulfatide antibodiesin the absence of a detectable serum M-protein may be greater thanpreviously suspected. Study of sera from other clinically similarpatients with otherwise idiopathic sensory or sensory plus motorneuropathies may uncover high anti-S-carb antibodies directed againstsulfatide or MAG. ELISA assays performed at 4° C., in the absence ofdetergent and using BSA in wash solutions are particularly sensitive forsuch testing (Pestronk et al., 1990, Ann. Neurol. 27:316-326; Marcus etal., 1989, J. Neuroimmunol. 25:255-259). Based on our experience, serumanti-S-carb antibodies are more likely to occur in patients with distalgreater than proximal polyneuropathies than in patients with sensoryganglioneuropathy with prominent early proximal or upper extremityinvolvement.

                  TABLE V                                                         ______________________________________                                        IgM versus                                                                      Pa-                                                                           tient Sulfa-                                                                  #  tide MAG P.sub.o Ch--S--A Ch--S--C GM1 MA1                               ______________________________________                                        3    232,350     0      0 0       0      0       0                              13  14,720  1,128 1,356 182 352 265   465                                     17  7,848  4,416   860 2,732 538 382 1,068                                    14  2,136  2,796 1,280 2,356 2,336 285 3,575                                  16    863 200,000    0 0 0 0    0                                           ______________________________________                                         Patterns of cross reactivity of antisulfatide and antiMAG sera with other     sulfated or neuropathyrelated antigens. P.sub.o = P.sub.o protein,            CH--S--A = Chondroitin sulfate A, CH--S--C = Chondroitin  # sulfate C, GM     = GM1 ganglioside, GA1 = asialoGM1 ganglioside. Titers were measured by       ELISA. Note that there is no relation between antibody titers to sulfatid     or MAG and those to the other antigens tested.                           

7. EXAMPLE: CHARACTERIZATION OF NEUROPROTEIN-1

7.1. PROTEIN IDENTIFICATION

Neuroprotein-1 (NP-1) is identified by gel chromatography and Westernblotting as 3 protein bands with approximate molecular weights of about36, 38 and 42 kD. The bands migrate between 32 and 47 kD molecularweight markers. NP-1 was specifically identified by its ability to bindto IgM antibodies from 3 sera (numbers W1160, W2333, and W2500). NP-1was enriched in the central nervous system (CNS) and spinal cordnon-myelin white matter. Many sera that react with sulfatide reactedwith this protein, but sera from some neuropathy patients that do notreact with sulfatide may react with NP-1. The 36-42 kD protein bandswere contained in a non-myelin human CNS pellet produced bycentrifugation at 34,000 rpm for 30 minutes in a discontinuous sucrosegradient with layers of 0.32M and 0.88M sucrose. Further purificationwas obtained after dilipidation in ether-ethanol (3:2) and washing threetimes in 1% Triton-X-100 by centrifuging at 10,000 rpm for 10-20minutes. After washing, the pellet from a 20,000 rpm centrifuge spin for30 minutes was separated by 12% polyacrylamide gel electrophoresis(PAGE). The specific protein bands, identified by appropriate molecularweight and antibody binding, were eluted from the gel and used forWestern blotting or ELISA assays. NP-1 was reactive with ricinhemagglutinin and peanut lectin. Thus NP-1 is presumably a glycoproteincontaining terminal Galβ1-3 GalNAc carbohydrate moieties.

7.2. PATIENT TESTING

We have identified over 70 patient sera with high titer (>1:1000)antibodies to this protein by ELISA testing. These patients generallyhave a mixed axonal and demyelinating sensory-motor polyneuropathy. Serafrom 20 control persons, 21 patients with ALS, and 15 patients withchronic inflammatory demyelinating polyneuropathy (CIDP) did not bind tosulfatide or to NP-1.

8. EXAMPLE: DIFFERENT REACTIVITY OF SERUM IgM TO GM1 GANGLIOSIDE ANDHISTONE H3 (NEUROPROTEIN-2) IN TREATABLE MULTIFOCAL MOTOR NEUROPATHY

8.1. MATERIALS AND METHODS

8.1.1. PATIENTS

We studied patients with motor system disorders or polyneuropathy andhigh serum titers of IgM anti-GM1 ganglioside antibodies. For this studyclinical syndromes were assigned to several categories. (1) Seventeenpatients had MMN with distal asymmetric weakness, no definite uppermotor neuron or bulbar signs, and motor conduction block onelectrodiagnostic testing (Pestronk et al., 1988, Ann. Neurol.24:73-78); Pestronk et al., 1990, Ann. Neurol. 27:316-326). These werefurther subdivided into a group of 9 patients who improved (withincreased strength of at least 1 grade on the MRC scale) after treatmentusing cyclophosphamide or chlorambucil. The remaining 8 patients withMMN were either untreated (6 patients) or had no improvement afterimmunosuppression (2 patients). (2) Twenty-five patients had distalasymmetric LMN signs, no definite evidence of bulbar or upper motorneuron involvement and only axonal changes on electrodiagnostic testing(Pestronk et al., 1990, 27:316-326). (3) Thirty-seven patients withclassic ALS were defined by previously reported criteria used to qualifypatients for a series of clinical treatment trials (Pestronk et al.,1988, Neurology 38:1457-1461). (4) Forty-one patients had sensory orsensory +motor peripheral polyneuropathies (PN). 5) Thirty unselectedsera from blood bank volunteers were used to obtain control values.

8.1.2. ANTIBODY ASSAYS

Sera were assayed for antibodies to purified GM1 ganglioside (Sigma) andto the 17 kD neural protein, later characterized as histone H3. Theprotein was purified from a non-myelin pellet (Norton et al., 1973, J.Neurochem. 21:1171-1191) of human CNS white matter. The pellet wasdilipidated with ether-ethanol (3:2), washed in 1% Triton-X-100, againin deionized water and finally in 0.1M Tris-HCl, 0.1M PMSF, 5 mM EGTA atpH 7.25. The pellet was then homogenized in ice-cold solubilizationbuffer (25 mM CHAPS, 2M NaCl, 1 mM EGTA, 0.15M Na₂ PO₄, 2% glycerol,0.1M PMSF, pH 7.25), incubated for 30 minutes at 4° C. and centrifugedat 100,000 g for 2 hours. The supernatant was then desalted,concentrated and subjected to preparative polyacrylamide gelelectrophoresis (PAGE). The specific bands were identified (afterWestern blotting (Pestronk et al., 1991, Neurology 41:357-362) of aparallel lane and staining with a human serum (W2393) that bindsstrongly to the protein) and eluted from the gel.

Our ELISA methodology has previously been described (Pestronk et al.,1990, Ann. Neurol. 27:316-326; Pestronk, 1990, Muscle & Nerve (in press)"Motor Neuropathies, Motor Neuron Disorders And AntiglycolipidAntibodies"). GM1 ganglioside (400 ng in 50 μl of methanol) was added towells and evaporated to dryness. The purified 17 kD protein (400 ng in100 μl of 0.01M phosphate-buffered saline (PBS) pH 7.2 with 0.15M NaCl)was added to wells and incubated overnight at 4° C. Any remainingbinding sites were blocked overnight at 4° C. with 100 μl of 1% humanserum albumin in PBS for IgM assays and 1% normal goat serum for IgGassays. Plates of the 17 kD protein but not GM1 ganglioside were thenwashed five times with 1% bovine serum albumin (BSA) and 0.05% Tween-20in PBS. Subsequent steps were performed at 4° C. Between steps washing(×5) was performed using PBS with 1% BSA without detergent. Serum wasdiluted in PBS with 1% BSA and added to wells for 5 hours. Antibodybinding to GM1 ganglioside or the 17 kD protein was measured usingovernight exposure to specific goat anti-human IgM or IgG linked tohorseradish peroxidase (organon Teknika-Cappel, West Chester, Pa.).Color was developed by adding substrate buffer (0.1M citrate buffer, pH4.5 with 0.004% H₂ O₂ and 0.1% phenylenediamine) until a standardpositive control reached an optical density (OD) of 0.6 above normalcontrols. Titers of antibodies were calculated from OD data byextrapolating readings to the OD that might be expected at a standarddilution of 1:100. In general, a serum antibody with a high titer of xwas detectable (>3 SD over negative controls) up to a dilution of atleast 1/x. High titers were more than 3 SD above the mean of a panel ofsera from blood bank volunteers. Values ≧350 units were high for serumIgM antibodies against GM1 ganglioside. Values ≧2000 were high for serumIgM antibodies against the 17 kD protein.

8.1.3. WESTERN BLOT

The non-myelin CNS pellet (10 μg of protein per lane) or purified NP-2(2 μg) were fractionated using 15% PAGE and transferred ontonitrocellulose sheets. Test serums were diluted 1:500-1:4000 in PBS with1% BSA and then incubated with nitrocellulose strips for 2 hours at roomtemperature. After washing ×5 using PBS with 1% BSA, the binding ofimmunoglobulin was detected using 1 hour exposure to goat anti-human IgMlinked to horseradish peroxidase in PBS with 1% BSA (working dilution,1:1000). Color was developed with 0.05% diaminobenzidine (DAB) and 0.01%H₂ O₂ in PBS.

8.2. RESULTS

8.2.1. WESTERN BLOTTING OF HIGH TITER ANTI-GM1 SERA

We initially tested high titer anti-GM1 ganglioside sera for binding toneural proteins by Western blot methodology (FIG. 6). High titeranti-GM1 ganglioside sera were grouped by patient diagnosis and testedat dilutions of 1:500 or more. IgM in ALS sera commonly boundselectively to a 17 kD protein present in CNS and peripheral nervehomogenates. Peripheral neuropathy (PN) sera also often demonstratedbinding to the 17 kD protein band. However, the pattern of binding ofIgM in neuropathy sera was frequently less selective than in ALS. MostPN sera reacted with at least one band in addition to the 17 kD protein.Only 20% of ALS sera showed binding to bands other than the 17 kDprotein. IgM reactivity in MMN sera tested never bound to the 17 kD bandand only rarely to others in CNS or peripheral nerve homogenates.

8.2.2. CHARACTERIZATION OF THE 17 kD PROTEIN

We attempted to purify the 17 kD protein to quantitate antibody bindingby ELISA and to obtain amino acid sequencing. We found that the 17 kDprotein was concentrated in the nonmyelin pellet of CNS white matter.Further purification was obtained by solubilization in high salt CHAPSbuffer (see methods). Preparative PAGE electrophoresis provided a finalisolation step. Sequencing of the first 30 amino acids showed almostcomplete homology with histone H3. Purification of histone H3 bystandard techniques from histone mixtures confirms that the 17 kDprotein is histone H3.

8.2.3. ELISA MEASUREMENT OF SERUM IgM REACTIVITY TO THE HISTONE H3(NEUROPROTEIN-2)

We used ELISA methodology to measure IGM antibody titers to histone H3(NP-2) in sera with high titers of IgM anti-GM1 ganglioside antibodies.In these sera the highest titers of anti-GM1 ganglioside antibodies(>7000) were generally in patients with MMN or LMN disorders (FIG. 7).There were significantly more (p<0.01) patients with anti-GM1ganglioside titers above 7000 in these groups than in ALS or PN groups.However, there was considerable overlap in titers among the diagnosticgroups.

In a panel of 30 unselected control sera from blood bank volunteers, themean titer of IgM against histone H3 (NP-2) was 243±468 (standarddeviation) units, with the mean plus 3 standard deviations (SD) at 1647units. In patient sera with high anti-GM1 ganglioside titers,anti-histone H3 (NP-2) titers varied from 0 to 75,160 (FIG. 8). Therewere significantly (p<0.001) more high titer sera in the ALS (81%;30/37) and PN groups (79%; 33/42) than in the MMN group (30%; 5/17).

The ratio of IgM titers to histone H3 (NP-2) and GM1 ganglioside foreach serum (histone H3 (NP-2):GM1 ganglioside ratio) provided the bestdistinction between MMN and other patient groups (FIG. 9). The medianhistone H3 (NP-2):GM1 ganglioside ratio for all sera tested was 2.37. Inindividual sera histone H3 (NP-2):GM1 ganglioside ratios varied greatly,from 0 to 47. There was no correlation between the absolute titer of IgManti-GM1 ganglioside antibodies in a serum and the histone H3 (NP-2):GM1ganglioside ratio. Patients with treatable MMN all had low ratios,ranging from 0 to 0.78. Overall, 82% (14/17) of MMN patients had ratiosbelow 0.79 (median=0.12). Patients with ALS and polyneuropathy hadsignificantly (p<0.0001) higher histone H3 (NP-2):GM1 gangliosideratios. The median ratio for ALS sera was 3.38 with only 14% (5/37)below 0.79. The median ratio for polyneuropathy sera was 3.02 with only7% (3/41) below 0.79. The overall statistics for the LMN group wereintermediate with a median ratio of 0.64 and 52% (13/25) below 0.79. Theintermediate value for the LMN group resulted from a large number ofhistone H3 (NP-2):GM1 ganglioside ratios with a value of 0 (36%; 9/25).If sera with ratios of 0 were deleted from the LMN and ALS groups, thenthe remaining populations of values were not significantly different.

8.3. DISCUSSION

8.3.1. FINE SPECIFICITIES OF ANTI-GM1 GANGLIOSIDE ANTIBODIES

Sera with high titers of antibodies to GM1 ganglioside may also reactwith other glycolipids or glycoproteins (Freddo et al., 1986, Neurology36:454-459; Pestronk et al., 1990, Ann. Neurol. 27:316-326; Shy et al.,1989, Ann. Neurol. 25:511-513; Latov et al., 1988, Neurology 38:763-768;Baba et al., 1989, J. Neuroimmunol. 25:143-150; Kusunoki et al., 1989,J. Neuroimmunol. 21:177-181; Nardelli et al., 1988, Ann. Neurol.23:524-528). The patterns of serum reactivity depend in part oninteractions of the antibodies with specific epitopes on thecarbohydrate or lipid moieties of GM1 ganglioside. Antibodies that reactwith the terminal disaccharide on GM1 ganglioside, Galβ1-3GalNAc, oftencross-react with other glycolipids that contain the same disaccharide,including asialo-GM1 and GD1b gangliosides. Antibodies with bindingproperties that involve the lipid moiety on GM1 ganglioside may reactwell with a wide spectrum of other glycolipids, but only poorly withglycoproteins (Chaudhry et al., 1990, Neurology 40:118S).

There is some data regarding the association of particular serum bindingpatterns with specific clinical syndromes. In MMN and LMN syndromes 3major fine specificities of anti-GM1 ganglioside antibodies have beendefined (Pestronk et al., 1990, Ann. Neurol. 27:316-326; Sadiq et al.,1990, Neurology 40:1067-1072; Baba et al., 1989, J. Neuroimmunol.25:143-150). Each of these reacts with precise carbohydrate epitopes onGM1 ganglioside. Changes in the terminal galactose of GM1 ganglioside,such as addition of a sialic acid, greatly reduce the binding ofanti-GM1 ganglioside antibodies from motor neuropathy and LMN patients.In contrast, the binding of antibodies that arise after immunizationwith GM1 ganglioside is less affected by changes in the carbohydratemoiety (Chaudhry et al., 1990, Neurology 40:118S).

The environment of the GM1 ganglioside molecule variably influences thebinding of anti-GM1 ganglioside antibodies in different disorders.Anti-GM1 ganglioside antibodies from patients with ALS generally bindwell to GM1 ganglioside in a lipid, membrane-like environment. However,antibodies from patients with MMN and LMN syndromes often do not reactwith GM1 ganglioside in membranes.

Despite the correlations between antibody specificity and clinicalsyndromes, the diagnostic and pathogenic role of anti-GM1 gangliosideantibodies require further investigation. There is evidence that someanti-GM1 ganglioside antibodies can bind to neural structures includingmotor neuron cell bodies and nerve terminals (Schluep et al., 1988,Neurology 38:1890-1892; Thomas et al., 1989, J. Neuroimmunol.23:167-174; Thomas et al., 1990, J. Neuropath Exp. Neurol. 49:89-95).Immunization of rabbits with GM1 ganglioside may induce neuropathy,possibly with motor conduction block (Nagai et al., 1976, Neurosci.Lett. 2:107-111; Thomas, et al., 1990, Ann. Neurology 28:238). However,it is important to explain why a range of neuropathy and motor neuronsyndromes are associated with anti-GM1 ganglioside antibody reactivity.Overlap in antibody binding patterns between diagnostic groups alsolimits the diagnostic utility of anti-GM1 ganglioside antibody testing.

8.3.2. REACTIVITY OF ANTI-GM1 GANGLIOSIDE SERA WITH HISTONE H3 (NP-2)

The results of our Western blot and ELISA testing show that the patternof serum IgM reactivity in MMN often differs from the patterns in ALSand polyneuropathy. Serum IgM from MMN patients generally reactsconsiderably more strongly with GM1 ganglioside than with histone H3(NP-2). Many MMN sera do not react with histone H3 (NP-2) at all. Most(82%) have histone H3 (NP-2):GM1 ganglioside ratios of less than 0.79.In contrast there is commonly high titer IgM reactivity to histone H3(NP-2) in anti-GM1 ganglioside sera from other patient groups. Some ALSand polyneuropathy sera react to histone H3 (NP-2) in titers that are 10to 40 times greater than those to GM1 ganglioside. Few (10%) havehistone H3 (NP-2):GM1 ganglioside ratios less than 0.79. Thus,measurement of histone H3 (NP-2):GM1 ganglioside ratios in high titeranti-GM1 ganglioside sera can increase specificity for MMN 10-fold. Alow histone H3 (NP-2):GM1 ganglioside ratio (<0.79) occurs in mostpatients with MMN, but 90% of anti-GM1 ganglioside sera from otherdisorders have high ratios. Further studies are necessary to determinewhether the patterns of IgM binding result from cross reactivity ofindividual IgM antibodies with both histone H3 (NP-2) and GM1ganglioside, or from the binding of different IgM molecules in the sameserum.

Our data suggest that definition of anti-GM1 ganglioside serumspecificities, and cross-reactivity with proteins such as histone H3(NP-2), is necessary before such results can be interpreted. For MMNautoantibodies with strong specificity for carbohydrate moieties on GM1ganglioside but with little reactivity to histone H3 (NP-2) should bestudied. For definition of the relation of anti-histone H3 (NP-2)antibodies to ALS and peripheral neuropathy, it will be important todetermine differences in serum reactivity between the two syndromes.Identification of an antibody binding pattern with some specificity forALS would likely provide a clue to the mechanism underlying thedisorder.

8.3.3. DIAGNOSTIC TESTING

The primary reason for the clinical measurement of anti-GM1 gangliosideantibodies is as a diagnostic aid in identifying MMN. This disorder isprobably immune-mediated and treatable (Pestronk et al., 1988, Ann.Neurol 24:73-78; Pestronk et al., 1990, Ann. Neurol. 27:316-326). MMNhas a therapeutic response profile of considerable improvement (in 80%of patients) in strength after treatment with sufficient doses ofcyclophosphamide. Prednisone is generally not effective.

High titers of anti-GM1 ganglioside antibodies occur in 60-80% ofpatients with MMN (Pestronk et al., 1990, Ann. Neurol. 27:316326).However, the diagnostic utility of anti-GM1 ganglioside testing islimited by the occurrence of these antibodies in 10-15% of patients withmore common disorders, including ALS and polyneuropathy. Our data nowshow that combined testing for antibodies to GM1 ganglioside and tohistone H3 (NP-2) provides a 5-10-fold increase in specificity withlittle reduction in sensitivity. Low histone H3 (NP-2):GM1 gangliosideantibody ratios represent in most MMN patients and in all those who haveresponded to cyclophosphamide but not to prednisone treatment. Themeaning of low histone H3 (NP-2):GM1 ganglioside ratios in otherdisorders with high titers of anti-GM1 ganglioside antibodies remains tobe determined. It will be especially interesting to compare the responseto different immunosuppressive regimens in LMN and neuropathy syndromeswith high or low histone H3 (NP-2):GM1 ganglioside ratios.

9. EXAMPLE: CHARACTERIZATION OF NEUROPROTEIN-3

9.1. PROTEIN IDENTIFICATION

β-Tubulin (neuroprotein-3 (NP-3)) has a molecular weight of 50-54 kD. Itmigrates on 12% PAGE just above the location of the Wolfgram proteins ina separation of human white matter or myelin. β-Tubulin (NP-3) isenriched in CNS myelin. It is specifically identified by the binding ofserum W1763. Purification was achieved from myelin prepared by themethod of Norton and Poduslo, 1973, J. Neurochem. 21:1171-191. CNSmyelin was dilipidated using a mixture of ether and ethanol at a ratioof 3:2, washed with 1% Triton-X-100. Pellets after each of these washeswere obtained by centrifuging at 10,000 rpm for 10-20minutes. The finalpellet was isolated, dissolved in 2 percent SDS, and then subjected topreparative electrophoresis on 12% PAGE. The specific protein waslocated on the gel by Western blotting with serum W1763 and molecularweight identification. The β-tubulin (NP-3) band was then eluted fromthe PAGE gel and concentrated.

Data suggests that β-tubulin (NP-3) may be highly homologous oridentical to beta-tubulin. The first 24 amino acid residues of β-tubulin(NP-3), depicted in FIG. 4 (SEQ ID NO:3), are strongly homologous withbeta-tubulin (SEQ ID NO:4), and serum W1763, which binds to NP-3, bindsto beta-tubulin. Further, monoclonal antibodies raised against NP-3react with beta-tubulin.

9.2. PATIENT TESTING

Patient sera were tested for antibodies against β-tubulin (NP-3) byWestern blotting and ELISA methodology. Normal values for levels ofantibodies against β-tubulin (NP-3) are less than 1:1000. We have testedover 60 sera from patients with inflammatory demyelinatingpolyneuropathies including Guillain-Barre and CIDP. Our results showthat 40% of patients with these disorders have IgM or IgG antibodies ina high titer against β-tubulin (NP-3). Testing of the same sera againstother glycolipids and glycoproteins including GM1 ganglioside, sulfatideand panels of neutral and acidic glycolipids in MAG show that less than5-10% have high titers of serum antibodies against other antigenictargets. Antibodies are present in high titer at the onset ofGuillain-Barre syndrome and fall over the course of the disease.

10. EXAMPLE: NEUROPROTEIN-4

Neuroprotein-4 (NP-4) has a molecular weight of approximately 20-24 kD.It migrates just above the large basic protein band on 15% PAGE. Theprotein is identified by binding with serum W1945. It was prepared bythe method as set forth for NP-2, including the washing in Tris buffer.The pellet was then dissolved in 2 percent SDS and subjected to PAGE ona 15 percent polyacrylamide gel. A protein band having a molecularweight of about 20-24 kD was identified. Western blots of serum from 14patients with polyneuropathies show that 5 patients have serum titers of1:500 or higher to NP-4.

11. EXAMPLE: NEUROPROTEIN-5

Neuroprotein-5 (NP-5) has a molecular weight of approximately 30-32 kD.It was prepared by differential centrifugation, washing, and elution ofspecific 30-32 kD bands from PAGE gels by methods similar to thoseutilized in the preparation of NP-4. It is identified by the binding ofserum 1.0286. NP-5 is present in peripheral nerve and non-myelin brainwhite matter.

12. EXAMPLE: CLINICAL SYNDROMES WITH SERUM ANTIBODIES TO SP NEURALANTIGEN, GM1 GANGLIOSIDE. AND SULFATIDE

12.1. ANTIGEN IDENTIFICATION

SP neural antigen and purified myelin-associated glycoprotein (MAG),substantially free of SP neural antigen, are obtained from partiallydelipidated, washed human central nervous system (CNS) myelin. SP neuralantigen is also referred to as central myelin antigen (CMA) and galopin.After solubilization of central nervous system (CNS) myelin in diluteZwittergent, SP neural antigen and its lipid components are precipitatedby centrifugation. Myelin proteins remain in the Zwittergent solution.Purified MAG is then separated from other myelin proteins bydifferential solubilization in Zwittergent:Phenol.

SP neural antigen is identified by reactivity with 2 sets of serums. Oneset of serums (numbers W110, 3.3197, GS, 2.1343 and 2.3604) is frompatients with motor neuropathies or lower motor neuron syndromes. Theseserums also usually react to GM1 ganglioside, and to components of SPneural antigen that migrate near GM1 ganglioside, using thin-layerchromatography (TLC), but have relatively low binding to histone H3. Asecond set of serums (numbers 2.1148, 2.1967, 2.0120, 2.2862, and2.3314) is from patients with sensory-motor neuropathies and, often,GALOP syndromes. These serums also usually react to sub-fractions ofsulfatide, and to components of SP neural antigen that migrate nearsulfatide sub-fractions, using TLC, but have little binding to GM1ganglioside. According to the present invention, the term "isolated andpurified" SP neural antigen means SP neural antigen substantially freeof myelin proteins, such as MAG, proteolipid protein and myelin basicprotein, as shown by Coomassie blue staining of polyacrylamide gels,Western blot and TLC. Coomassie blue staining of polyacrylamide gelsshows that the SP neural antigen preparation is substantially free ofall myelin proteins. Serums that are reactive by ELISA to SP neuralantigen from patients with GALOP syndromes, ataxic polyneuropathies ormotor syndromes generally do not react with MAG.

Analysis of TLC separations of isolated and purified SP neural antigenshow 3 major lipid fractions. (1) One fraction, that migrates nearest tothe solvent front, in regions usually associated with non-polar lipids,is not antigenic. (2) Serums from GALOP patients, and those withsensory-motor neuropathies, show a major antigenic peak migrating on TLCwith a reference value of 0.6 to 0.8, near commercial sulfatidestandards. Known positive serum number 1.2391 stains this second groupof highly purified SP neural antigen components but does not react, byELISA, with commercially prepared sulfatide that contains a mixture ofsulfatide sub-fractions that differ in the size and hydroxylation stateof their lipid moieties. Mass spectroscopy suggests that the SP neuralantigen components are more highly purified subfractions of sulfatidethan are present in commercial sulfatide. The whole SP neural antigenpreparation may be more antigenic (binds serum antibodies in highertiter) than the purified SP subfractions (exemplified by known positiveserum number 1.2391). (3) Serums from patients with motor neuropathies,and lower motor neuron syndromes, show a major antigenic peak migratingon TLC with a reference value of 0.3 to 0.5, near commercial GM1ganglioside standards. These SP neural antigen components havepreviously been referred to as NP-9 antigens. The whole SP neuralantigen preparation is often more antigenic than the isolated NP-9antigen.

SP neural antigen was initially isolated from CNS myelin by lithiumdiiodosalicylate (LIS) methodology as previously described for MAG(Quarles, 1977, Biochem. J., 163:635-637; Quarles et al., 1983, Biochim.Biophys. Acta, 757:140-143). We have developed a new method thatsubstantially separates SP neural antigen from MAG by replacing the LISwith Zwittergent. This produces a highly purified preparation of MAGthat can be used for the specific identification of serum anti-MAGantibodies by ELISA methodology. The MAG preparations now in use containimpurities and produce many false positive ELISA results. Thezwittergent method also produces purified SP neural antigen that can beused to screen serums for antibodies to NP-9 antigen reactivity and theantibody reactivity found in GALOP and sensory-motor neuropathypatients.

Myelin is purified from human central nervous system white matter bysucrose gradient methodology. White matter is homogenized is 0.88Msucrose, and 0.32M sucrose is layered on top of the homogenate. Thematerial is then centrifuged at about 42,000 rpm. Myelin is obtainedfrom the interface between the sucrose concentrations, diluted in colddeionized water and centrifuged at about 14,000 rpm for about 15minutes. The resulting pellet is resuspended in deionized water,homogenized and centrifuged at about 10,000 rpm for about 10 minutes.Next, the sucrose gradient and washing procedures are repeated. Themyelin is resuspended in a small amount of water and frozen at about-80° C. Lyophilization is then carried out at a vacuum of about <100millitorr at about 23° C.

Substantially purified SP neural antigen and MAG antigens according tothe invention are prepared by a process which comprises:

a) partially delipidating CNS myelin using a solvent;

b) suspending impure SP neural antigen in a second solvent;

c) centrifuging to separate the SP neural antigen (in the pellet) fromMAG and other myelin components;

d) solubilizing the SP neural antigen in the pellet in a third solvent;

d) adding a fourth solvent to the MAG-containing supernatant andcentrifuging to separate substantially purified MAG in one solvent phasefrom other myelin proteins in the second phase.

In a preferred embodiment of the invention, multiple sequentialcentrifugations are repeated with intermittent washings to preparesubstantially purified SP neural antigen and MAG. The components of SPneural antigen may be further purified by TLC to yield isolatedfractions substantially free of all myelin proteins and many myelinlipids. It should be recognized that other procedures may be employed toyield purified SP neural antigen and its components; particularlypreferred procedures are set forth below in the following examples.

Patient sera have been deposited with the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A.

The following examples are given to illustrate the invention but are notdeemed to be limiting thereof.

12.1.1. EXAMPLE

This example illustrates the preparation of isolated and purified SPneural antigen according to the invention, and the utilization andinterpretation of results of antibody assays.

The following process is used to prepare isolated, purified SP neuralantigen and MAG:

a) suspending about 1 to 2.4 g of lyophilized myelin inhexane:2-propanol (3:2, v/v) at a concentration of about 180 ml per 1 gdry weight of myelin.

b) stirring the suspension at room temperature (22° C.) for about 30minutes;

c) centrifuging the suspension for about 30 minutes at 19,000 rpm(50,000 g)

d) washing the residue with diethyl ether;

e) centrifuging the diethyl ether-solubilized residue for about 15minutes at about 6,000 rpm;

f) drying the diethyl ether-solubilized residue under nitrogen;

g) suspending the residue in 0.05M Tris/HCl containing 2.5 to 7.5 mMZwittergent (using a Dounce homogenizer or a motorized homogenizer),with 1.5 ml Tris/HCl--Zwittergent for each 50 mg dry weight oflyophilized myelin starting material;

h) stirring the suspension overnight in a cold room (4° C.);

i) adding 1.5 volumes of water and centrifuging for about 30 minutes atabout 26,000 rpm (78,000 g).

At this point in the preparative process the pellet contains the SPneural antigen and MAG is present in the supernatant.

The SP neural antigen is further purified by:

j) adding methanol (1 ml per 25 mg dry weight of lyophilized myelinstarting material) to the pellet;

k) suspending the pellet using a vortex or polytron;

l) centrifuging the suspension for about 15 minutes at about 6,000 rpm;

m) drying the material (SP neural antigen) that remains suspended orsolubilized;

n) titering the SP neural antigen by running an ELISA assay usingdifferent concentrations of SP neural antigen per well and knownpositive serums (for example, serums #2.1148, #2.1967, #2.0120, #2.2862,#2.3314, GS or #1.0762) and negative serum.

Purified subfractions of lipids in SP neural antigen are obtained by:

o) spotting SP neural antigen on a silica high performance TLC (HPTLC)plate;

p) separating lipids using solvents, including chloroform: methanol:0.2% CaCl₂ in water;

q) purified antigens are removed from the HPTLC plate by scraping bandsof silica from the plate and washing several times with solvents, suchas chloroform: methanol and water. These antigens are substantially freeof myelin proteins;

r) identifying and measuring amounts of purified antigens by ELISAassays using serum #2.1148, #2.1967, #2.0121, #2.2862, or #2.3314 toidentify antigens related to GALOP syndromes (these usually migrate nearsulfatide standards) and serum #W110, #3.3197, GS, #2.1343 and #2.3604to identify antigens related to motor syndromes and NP-9 antigenreactivity (these usually migrate near GM1 standards).

MAG is further purified from the supernatant obtained in step i) by:

s) adding an equal volume of 50% phenol to the supernatant;

t) stirring in a cold room (4° C.) for 30 minutes;

u) centrifuging at 6,000 rpm (4,000×g) for 45 minutes;

v) allowing the mixture to stand until 2 phases form;

w) removing and dialyzing the upper phase exhaustively with water toremove the phenol and Zwittergent;

x) clarifying the MAG by ultracentrifugation at 29,000 rpm (100,000 g);

y) lyophilizing the supernatant; and

z) identifying and measuring the MAG by ELISA assay using serum numbers#W1177, #2.2705, and #2.3200;

Synthetic SP neural antigen is prepared by:

aa) combining GM1 ganglioside, sulfatide, and cholesterol (1: 10: 10 byweight) in methanol;

bb) drying the mixture; and

cc) resuspending in phosphate-buffered saline

Clinical utilization of SP neural antigen and MAG and interpretation ofantibody binding assays involves several steps, including:

dd) measuring the presence, and levels, of binding of IgM and IgG frompatient serums, using ELISA assays (and Western blots, whereappropriate), to SP neural antigen, MAG, GM1 ganglioside, sulfatide,purified fractions of SP neural antigen, or synthetic SP neural antigen;

ee) comparing levels of serum antibody binding to histone H3. Serumswith much lower binding to histone H3 than to one or more of theantigens in aa) are interpreted as having specific antibody reactivityand relatively strong correlations with specific clinical syndromes.Serums with similar or higher binding to histone H3 relative to one ofthe antigens in aa) are considered relatively polyreactive and with lessstrong correlations to specific clinical syndromes;

ff) comparing titers of specific serum antibody binding to the SP neuralantigen to the antibody binding to other antigens listed in dd). Serumswith high titers of antibody binding to SP neural antigen alonecorrelate with syndromes having sensory (±motor) signs and often gaitdisorders (including GALOP syndromes). Serums with binding both to SPneural antigen and to sulfatide (or to purified SP neural antigensubfractions migrating on TLC near sulfatide) also correlate withsyndromes having sensory (±motor) signs and often gait disorders(including GALOP syndromes). Serums with binding both to SP neuralantigen and to GM1 ganglioside are designated as having NP-9 antigenreactivity and correlate with motor neuropathies and lower motor neuronsyndromes. Serums with very high specific binding to GM1 ganglioside,but much lower binding to SP neural antigen, also correlate with motorneuropathies and lower motor neuron syndromes;

gg) comparing titers of specific antibody binding to MAG, assayed byELISA, with binding to MAG by Western blot. Serums with binding to MAGon both assays correlate with polyneuropathies with sensory motor) signsand evidence of demyelination. Serums with relatively low ELISA titersof binding to MAG (<1:6,000), or binding to MAG by ELISA but not byWestern blot correlate with polyneuropathies, but the specific clinicalfeatures are less predictable or stereotyped.

12.2. ANTIBODY ASSAYS

Serums were assayed for IgM binding to proteins and glycolipids by ELISAmethodology. Pure lipid antigens (e.g. 500 ng of sulfatide dissolved in15 μl of methanol), were added to wells and evaporated to dryness. SPneural antigen and protein antigens (50 to 2000 ng in 100 μl of 0.01Mphosphate-buffered saline (PBS) pH 7.2 with 0.15M NaCl) were added towells and incubated overnight at 4° C. For both protein and lipidantigens, remaining binding sites were blocked with 100 μl of 1% humanserum albumin in PBS for 4 hours, at room temperature. Plates were thenwashed three times with 1% bovine serum albumin (BSA) (and 0.05%Tween-20 for protein antigens) in PBS. Subsequent steps were performedat 4° C. Between steps, washing (×3) was performed using PBS with 1% BSAwithout detergent. All serums were tested by adding 100 μl of dilutions(1:1,000 to 1:300,000 in PBS with 1% BSA) in duplicate to wellsovernight, then washing with PBS. The binding of IgM to antigens wasmeasured using 4-hour exposure to goat anti-human IgM linked tohorseradish peroxidase (HRP) (organon Teknika-Cappel, West Chester, Pa.)in PBS with 1% BSA (1:20,000). Color was developed with 100 μl substratebuffer (0.1M citrate buffer, pH 4.5, with 0.004% H₂ O₂ and 0.1%phenylenediamine) for 30 minutes. Optical density (OD) was determined at450 nm. A serum antibody with a titer of x was detectable (>0.05 ODunits over controls) up to a dilution of at least 1/x. Antibody titersof greater than 2,500 were positive.

Patients with GALOP syndromes had antibody titers greater than 10,000,no binding to histone H3 at titers ≧35% of those to SP neural antigen,and no specific binding to GM1 ganglioside. Patients with motorsyndromes had titers of antibody binding to SP neural antigen ≧2,500 andhigh titers of antibody binding to GM1 ganglioside, but lower binding tohistone H3.

SP neural antigen (also called referred to as central myelin antigen(CMA) and galopin) a purified combination of polar lipids from the humancentral nervous system, freed of myelin associated proteins by serialsteps of differential solubilization and centrifugation, andspecifically identified by enzyme-linked immunosorbent assays of wholeSP neural antigen using know positive serums #2.1148, #2.1967, #2.0120,#2.2862, and #2.3314 from GALOP patients and numbers #W110, #1.0762,#3.3197, GS, #2.1343 and #2.3604 from motor neuropathy patients.

SP neural antigen can be further characterized according to its lipidcomponents by thin layer chromatography using solvents, includingchloroform: methanol: water and chloroform: methanol: CaCl₂ in water.

Purified and isolated SP neural antigen can be prepared by a preferredprocess comprising the steps of:

a) suspending about 1 to 2.4 g of lyophilized myelin inhexane:2-propanol (3:2, v/v) at a concentration of about 180 ml per 1 gdry weight of myelin.

b) stirring the suspension at room temperature (22° C.) for about 30minutes;

c) centrifuging the suspension for about 30 minutes at 19,000 rpm(50,000 g)

d) washing the residue with diethyl ether;

e) centrifuging the diethyl ether-solubilized residue for about 15minutes at about 6,000 rpm;

f) drying the diethyl ether-solubilized residue under nitrogen;

g) suspending the residue in 0.05M Tris/HCl containing 2.5 to 7.5 mMZwittergent (using a Dounce homogenizer or a motorized homogenizer),with 1.5 ml Tris/HCl--Zwittergent for each 50 mg dry weight oflyophilized myelin starting material;

h) stirring the suspension overnight in a cold room (4° C.);

i) adding 1.5 volumes of water and centrifuging for about 30 minutes atabout 26,000 rpm (78,000 g);

j) adding methanol (1 ml per 25 mg dry weight of lyophilized myelinstarting material) to the pellet;

k) suspending the pellet using a vortex or polytron;

l) centrifuging the suspension for about 15 minutes at about 6,000 rpm;

m) drying the material (SP neural antigen) that remains suspended orsolubilized; and

n) titering the SP neural antigen by running an ELISA using differentconcentrations of SP neural antigen per well and known positive serums(for example, serum #2.1148, #2.1967, #2.0120, #2.2862 or #2.3314) andnegative serum.

The purified subfractions of purified SP neural antigen can be furtherseparated by thin layer chromatography using solvents (such aschloroform: methanol: water or chloroform: methanol: CaCl₂ in water) andrecovered from the chromatography plate by single, or repeated, washingof sequential regions of the TLC plate. Such a TLC separation can becarried out in a preferred embodiment of the present invention by:

o) spotting SP neural antigen on a silica high performance TLC (HPTLC)plate;

p) separating lipids using solvents, including chloroform: methanol:0.2% CaCl₂ in water;

q) removing purified antigens from the HPTLC plate by scraping bands ofsilica from the plate and washing several times with solvents, such aschloroform/methanol and water. These antigens are substantially free ofmyelin proteins; and

r) identifying and measuring amounts of the purified SP neural antigenby ELISA assays using sera #2.1148, #2.1967, #2.0121, #2.2862, or#2.3314 to identify antigens related to GALOP syndromes (these usuallymigrate near sulfatide standards) and serA #W110, #3.3197, GS, #2.1343and #2.3604 to identify antigens related to motor syndromes and NP-9reactivity (these usually migrate near GM1 ganglioside standards).

In a preferred embodiment of the present invention, synthetic SP neuralantigen can be prepared by combining lipid components, including GM1ganglioside, sulfatide, and cholesterol (for example, 1:10:10 byweight). Other lipid components, such as galactocerebroside (preferablywith a non-hydroxylated fatty acid moiety), may also be added to improveantigenicity or specificity.

Purified myelin-associated glycoprotein (MAG), substantially free of SPneural antigen, can be prepared by a preferred process comprising thesteps of:

s) adding an equal volume of 50% phenol to the supernatant in step l)above;

t) stirring in a cold room (4° C.) for 30 minutes;

u) centrifuging at 6,000 rpm (4,000 g) for 45 minutes;

v) allowing the mixture to stand until 2 phases form;

w) removing and dialyzing the upper phase exhaustively with water toremove the phenol and Zwittergent;

x) clarifying the MAG by ultracentrifugation at 29,000 rpm (100,000 g);

y) lyophilizing the supernatant; and

z) identifying and measuring the MAG by ELISA assay using sera #W1177,#2.2705, and #2.3200.

The semipurified, purified, and synthetic SP neural antigen can besolubilized or suspended in solvents, including methanol andphosphate-buffered saline for use as antigens in immunoassays. Thesematerials may further comprise lyophilized and washed proteins andlipids.

Another aspect of the present invention is to provide a method ofdiagnosing a gait disorder and neuropathy in a patient comprising thesteps of: mixing a patient's serum, or the IgM or IgG in the serum, withSP neural antigen, and determining the titer of serum IgM or IgGantibodies that bind to SP neural antigen, wherein a titer of IgM or IgGantibodies to SP neural antigen of at least 1:10,000 correlatespositively with a syndrome of gait disorder and neuropathy.

In another preferred embodiment, a method of diagnosing a syndrome of agait disorder and neuropathy in a patient comprises the steps of: mixinga patient's serum, or the IgM or IgG in the serum, with, individually,GM1 ganglioside, histone H3, sulfatide, and SP neural antigen;determining the titer of serum antibodies that bind to GM1 ganglioside;determining the titer of serum antibodies that bind to histone H3;determining the titer of serum antibodies that bind to sulfatide; anddetermining the titer of serum antibodies that bind to SP neuralantigen; wherein a titer of serum antibodies to SP neural antigen of atleast about 10,000, no specific binding to GM1 ganglioside, and nobinding to histone H3 at titers ≧35% of the binding to SP neural antigencorrelates positively with a syndrome of gait disorder and neuropathy.

Still further, the present invention provides a method of diagnosing asyndrome of gait disorder and neuropathy comprising the steps of mixingthe patient's serum sample with the lipid subfractions of SP neuralantigen, and determining the titer of antibodies that bind to thesesubfractions.

These diagnostic methods are effective in diagnosing clinical syndromeswhich are predominantly sensory neuropathies and sensory-motorneuropathies. In particular, a triad of serum IgM antibody reactivity toSP neural antigen (serum antibody titer greater than about 2,000), toGM1 ganglioside (serum antibody titer greater than about 600) and tohistone H3 (serum antibody titer less than about 0.62 times the serumIgM antibody titer to GM1 ganglioside) correlates positively with apredominantly motor syndrome without spasticity. The positivecorrelation is particularly apparent where the predominantly motorsyndrome without spasticity is a multifocal motor neuropathy and isstill more apparent where the multifocal motor neuropathy has a clinicalhistory of asymmetric motor weakness with electrophysiological evidenceof motor conduction block, with or without axonal loss. The positivecorrelation is observed where the predominantly motor syndrome is apredominantly distal lower motor neuron disorder and, more particularly,where the distal lower motor syndrome has a history of distal asymmetricweakness which begins in a hand and foot.

The SP neural antigen of the present invention can be furthercharacterized by high degrees of binding of IgM antibodies to SP neuralantigen in patient sera #1.0421 and #2.0328 (serum IgM antibody titergreater than about 1:2,500) and by a relatively low degree of binding ofIgM antibodies to SP neural antigen in patient serum #2.1343 compared tothe binding of IgM in this serum to GM1 ganglioside.

In another preferred embodiment of the present invention, a method ofdiagnosing a predominantly motor syndrome in a patient comprises thesteps of determining in a serum sample from a patient the titer of IgMantibodies that bind to GM1 ganglioside, to SP neural antigen, and tohistone H3; wherein a titer of IgM antibodies binding to SP neuralantigen greater than about 2,000, a titer of IgM antibodies binding toGM1 ganglioside greater than about 600, and a relative titer of IgMantibodies binding to histone H3 less than about 0.62 times the titer ofIgM antibodies binding to GM1 ganglioside correlates positively with apredominantly motor syndrome without spasticity. This positivecorrelation is particularly apparent where the predominantly motorsyndrome without spasticity is a multifocal motor neuropathy and isstill more apparent where the multifocal motor neuropathy has a clinicalhistory of asymmetric motor weakness with electrophysiological evidenceof motor conduction block, with or without axonal loss. The positivecorrelation is observed where the predominantly motor syndrome is apredominantly distal lower motor neuron disorder and, more particularly,where the distal lower motor syndrome has a history of distal asymmetricweakness which begins in a hand and foot.

The semipurified, purified, or synthetic SP neural antigen can beemployed in a kit for diagnosing neuropathy, either with or without gaitdisorder. Such a kit, in a preferred embodiment of the present inventioncan comprise substantially purified, or synthetic, SP neural antigen;lipid subfractions of SP neural antigen; histone H3; and detectablylabeled antibody that binds to human antibody directed against the SPneural antigen.

Another aspect of the present invention provides a method of producingan animal model system for neuropathy which comprises immunizing ananimal with SP neural antigen and an immune adjuvant. In a preferredembodiment, the SP neural antigen comprises purified subfractions of SPneural antigen.

The SP neural antigen of the present invention can be used to provide asubstantially purified antibody that binds to SP neural antigen and,more preferably, the antibody is a monoclonal antibody.

Additionally, the semipurified, purified, or synthetic SP neural antigenof the present invention can be employed in a kit for diagnosing aneuropathy, which comprises substantially purified, or synthetic, SPneural antigen; substantially purified GM1 ganglioside antigen;substantially purified histone H3 antigen; and detectably labeledantibody that binds to human IgM antibody.

The purified MAG of the present invention can be employed in a method ofdiagnosing a neuropathy in a patient which comprises the steps of mixingserum samples of a patient with the purified MAG of the presentinvention (that is substantially free of SP neural antigen); determiningthe titer of IgM antibodies binding to MAG; and determining the titer ofIgM antibodies binding to histone H3; wherein a titer of serumantibodies binding to MAG of at least about 1:2,000, and no binding tohistone H3 at titers above 55% of the binding to MAG correlatespositively with a neuropathy. This positive correlation is particularlyapparent where the neuropathy is a predominantly sensory neuropathy and,more particularly, where the neuropathy is a sensory-motor neuropathy.This positive correlation is most particularly apparent where theneuropathy is associated with electrophysiological evidence ofdemyelination, with, or without, evidence of axonal loss.

Additionally, the purified MAG, substantially free of SP neural antigen,of the present invention can be employed in a kit for diagnosing aneuropathy, which comprises substantially purified MAG that issubstantially free of SP neural antigen; histone H3; and detectablylabeled antibody that binds to human antibody directed against MAG.

12.3 PATIENT TESTING

Sera from GALOP syndrome patients as well as from 141 control patients;36 who were normal, 18 who had sensory ganglionopathies, 25 who hadAlzheimer's disease, 32 who had amyotrophic lateral sclerosis, and 30who had polyneuropathy, were evaluated by enzyme-linked immunosorbentassay (ELISA). Seven hundred twenty two other sera from samplessubmitted to our laboratory for evaluation were also tested.

Strength testing was quantitated using a hand-held dynamometer(Chatillon, Kew Gardens, N.Y.). Values determined on testing wereexpressed as pounds of resistance.

The findings in 7 patients, 6 patients with GALOP syndrome and 1 patientwith high titers of IgM binding to galopin and a mild polyneuropathy butwithout a gait disorder revealed that, by ELISA assay, all 6 GALOPsyndrome patients had extremely high titers of serum IgM binding to ahigh molecular weight antigen from CNS myelin (galopin (GRA)). In 4patients, serum IgM also reacted strongly to sulfatide. In 1 patient, ahigh titer of serum IgM reactive to MAG was found. The other 715 seratested during this period had titers of specific IgM binding to galopinthat were <10,000.

The six patients with GALOP syndrome averaged 73 years of age at thetime of diagnosis. All had a disabling gait disorder that was slowlyprogressive over 2-15 years, and polyneuropathy with sensory loss,involving all modalities, localized distally in the legs. Three patientshad findings consistent with a predominantly demyelinating neuropathy.One had only evidence of axonal loss. The five patients who were treatedhad clear improvement in ambulation. For example, patient 1 no longerneeded canes to ambulate and patient 5 was able to walk independentlyfor the first time in several years.

Patient 1, an 80-year old female, was evaluated for a progressive gaitdisorder. She felt numbness and tingling in her toes 4 years prior toevaluation. One year later she suffered from unsteadiness of gait andfalling. These symptoms gradually progressed until, at the time ofevaluation, numbness and tingling had advanced proximally to themid-calf level and mild weakness in the ankles had developed. Fallingoccurred as frequently as once per week, and the patient used two canesto walk distances greater than 5-10 feet. General examination revealedmultiple skin bruises and senile purpura. Neurological examinationshowed normal strength other than mild (4 to 4+ out of 5) weakness atthe ankles, ankle dorsiflexor strength averaged 32 pounds, and reflexeswere absent at the ankles but 2+ elsewhere. Pain, light touch,temperature and vibratory sensation were reduced markedly in the legs upto the knee and moderately in the arms to the mid forearm. Jointposition sense was moderately impaired in the toes but preservedelsewhere, and finger-nose and heel-shin testing was unremarkable. Thepatient's gait was shuffling with small steps. She could not walk morethan 10 feet without support, walk on her heels or toes, or performtandem walking. She had particular difficulty with turning or standingstationary with her feet together. The Romberg test was positive.Electrodiagnostic testing revealed low normal orthodromic sensory nerveaction potentials (SNAPS) amplitudes in the arms (5-8 μV) withmoderately slowed conduction velocities (38-40 m/s). SNAPs were absentin the legs. Motor studies showed borderline-low compound muscle actionpotential (CMAPS) and conduction velocities in the legs (1.3-5 mV; 38-40m/s) and normal CMAPs in the arms.

The studies were interpreted as being consistent with a moderate axonalpolyneuropathy affecting the legs, and the clinical impression was thatpatient 1 had a moderately severe sensory-motor polyneuropathy. However,the patient's gait disorder and loss of balance were much more severethan could be explained by the polyneuropathy alone.

In patient 1, the titer of serum IgM to galopin (GRA) was 16,400. IgM inthis serum also reacted in high titer (10,000) to sulfatide but not toother antigens tested, including GM1 ganglioside, asialo-GM1, GT1b, andmyelin-associated glycoprotein (MAG). Patient 1 was treated withintravenous human immunoglobulin (IV Ig) (1 g/kg/day×2 days). Distalstrength had improved on examinations 1 and 3 months later. Ankledorsiflexion strength averaged 42 pounds, joint position sense wasnormal, other sensory modalities were reduced up to the lower third ofthe calf, the patient could walk in tandem and on her heels and toes,and the Romberg test was negative. After 5 months, gait instabilityreturned and the patient began to use a cane to aid ambulation. Therewas no change in antibody titers after IV Ig treatment. Mosthematological and biochemical screening studies, including vitamins B₁₂and E and cryoglobulins were normal. Anti-nuclear antibody (ANA) was1:640. Total IgM and IgG were 424 (normal is less than 355) and 3200(normal is less than 1830), respectively. Serum and urine immunofixationwere normal.

Patient 2, a 62-year old female, was evaluated for increasing difficultywith walking and balance over 1 year. She became unable to bowl or playtennis, walking was reduced from 8-10 miles to 1-2 miles per week, anddifficulty with stairs developed. She noticed numbness and electricfeelings in her legs in the months prior to evaluation. Generalexamination was unremarkable. Neurological examination revealed normalstrength other than distal weakness (4 to 4+ out of 5) at the ankles.Ankle dorsiflexion strength averaged 12 pounds, reflexes were absent,pain and temperature sensations were reduced to the knee, and vibrationwas absent in the toes and reduced at the ankles. Joint position wasslightly reduced in the toes but preserved elsewhere, finger-nose andheel-shin testing was unremarkable, the patient could not walk intandem, and the Romberg test was positive. The patient's gait wasunsteady with a variable base. Electrodiagnostic testing in the legsrevealed normal SNAP amplitudes (7-15 mV) and borderline-low CMAPamplitudes (0.9-3.5 mV), but reduced conduction velocities (22-28 m/s)with prolonged distal latencies (11-16 m/s). In her arms, CMAPamplitudes were normal, conduction velocities were borderline reduced(43-44 m/s) and terminal latencies prolonged (6.8-8.5 m/s).

The results were interpreted as consistent with a mixed axonal anddemyelinating polyneuropathy affecting the legs more than the arms. Theclinical impression was that patient 2 had a mild to moderatepolyneuropathy, however, the gait disorder was more severe than could beexplained by the polyneuropathy alone.

In patient 2, the IgM titer to galopin (GRA) was 110,000. There was adegree of IgM binding to MAG (titer=6000) and to GT1b (titer=3000), butnot to GM1, asialo-GM1, or sulfatide. Prednisone (40 mg qd) and IV Ig (2g/kg) produced no improvement in the patient's condition. She thenreceived 7 monthly treatments of plasmapheresis on 2 successive daysfollowed by intravenous cyclophosphamide (1 gM/M²) . After 7 months ofplasma exchange and cyclophosphamide treatment, the titer of serum IgMto galopin (GPA) was 54,000. Examination after the final treatmentshowed improved distal strength. Ankle dorsiflexion strength averaged 24pounds, joint position sense was slightly reduced in the toes, othersensor modalities were reduced up to the ankles, and gait was unsteady.Functional testing showed improved ability to walk distances and climbstairs. Patient 2 could walk 2 steps in tandem. Most hematological andbiochemical studies, including vitamins B₁₂ and cryoglobulins, werenormal. ANA was positive at 1:2560. Sedimentation rate was 43. Serumimmunofixation showed an IgM paraprotein. Quantitative immunoglobulinswere normal. Patients 3, 4, and 6 had high serum titers of IgM vssulfatide but not to other glycolipid or glycoprotein antigens tested.

In the 722 other sera subjected to diagnostic testing, 151 serum wasidentified (patient 7) with very high, selective titer (>10,000) IgMreactivity to galopin (GRA). This 79-year old patient had a distalsensory neuropathy associated with an IgM lambda M-protein. The mildgait disorder in patient 7 was attributed to pain on the plantar surfaceof the feet.

No high titer anti-galopin binding was identified in the 141 sera fromdefined controls.

12.4. RESULTS

The 6 Galop syndrome patients developed a gait disorder over a period of2-15 years involving slowed ambulation and frequent falling, oftenbackwards. Examination revealed an uncertain gait with small steps anddifficulty with turning. This pattern of ambulation clinically resemblesmotor disorders noted with increasing age (Drachman et al., 1984,Clinical Neurology of Aging, Oxford University Press, Martin L. Albert,ed., pp. 97-113; Sudarsky, "Geriatrics: Gait Disorders in the Elderly,"NEJM, 1990, 322:1441-1446). None of the specific system findings,however, could entirely explain the degree of gait disability. Overall,the anatomical localization of the lesion(s) responsible for the gaitdifficulty remain undefined. The sensory loss and motor changes probablycontributed to the gait disorder.

GALOP syndrome appears to be treatable and so should be considered as acomponent of the differential diagnosis in gait disorders of theelderly. Several lines of evidence suggest that GALOP syndrome may beimmune-mediated. Two patients had serum ANA in high titer. Three had anIgM M-protein and three a polyclonal elevation in IgM levels. All hadvery high titers of serum IgM against a high molecular weight antigen,galopin (GRA). The improvement of 5 patients after immunomodulatingtherapy further evidences an immune etiology for Galop syndrome.

Very high titers of IgM autoantibodies to galopin (GRA) (>1:10,000) arespecific for GALOP syndrome (FIG. 10). Such high titers have been foundin only 1 of 722 sera submitted for testing, and in none of the 141 serafrom well-defined normal and neurologic controls, evidencing that thespecificity of antigalopin antibodies for GALOP syndrome andpolyneuropathy is high.

13. EXAMPLE: MOTOR NEUROPATHY SYNDROME ASSOCIATED WITH SERUM ANTIBODIESTO GM1 GANGLIOSIDE, AND NP-9 ANTIGEN, BUT NOT TO HISTONE H3

13.1. PROTEIN IDENTIFICATION

13.1.1. HISTONE H3

Histone H3 has a molecular weight of approximately 15,117 daltons. Itwas purified by purchasing the f₃ fraction of histones from a commercialsupplier and subjecting it to electrophoresis on 7.5% PAGE. Theappropriate 15-17 kD bands were identified by monoclonal antibody 5H10and patient serum W2393, and the protein is eluted from the PAGE gel.N-terminal amino acid sequencing of the N-terminal sequence of the 15-17KD protein (FIG. 3) (SEQ ID No:1) has confirmed that the antigenicprotein is histone H3 (SEQ ID NO:2).

Histone H3 of the present invention can be employed to construct anantibody that binds to histone H3 and, more preferably, the antibody isa monoclonal antibody.

13.1.2. NP-9 ANTIGEN

Partially purified NP-9 antigen copurifies with myelin glycoproteins,such as myelin-associated glycoprotein (MAG). Purified NP-9 antigenmigrates on thin layer chromatogram (TLC) as a polar lipid.Immunostaining of a TLC separation of NP-9 antigen from semipure NP-9antigen shows a spot by patient serum GS between about 1.9 and 2.1 cmfrom the origin. NP-9 antigen was prepared from CNS myelin by serialdifferential solubility steps. Myelin was prepared by the method ofNorton and Poduslo, 1973, J. Neurochem. 21: 1171-1191, as set forth forNP-3. About 1 to 1.2 g lyophilized myelin was suspended inchloroform/methanol (2:1, v/v) and centrifuged. Alternatively, themyelin can be suspended in hexane:2-propanol (3:2, v/v). The resultingpellet was washed with diethyl ether, centrifuged, and dried undernitrogen. Then the residue was dispersed in 0.05M Tris/HCl containing0.08M recrystallized lithium 3,5 diiodosalicylate (LIS). The suspensionwas stirred in a cold room overnight, then centrifuged. The supernatantwas retained as an enriched source of MAG. The pellet was dispersed in0.05M Tris/HCl containing 0.3M LIS. The suspension was stirred in a coldroom overnight, then centrifuged.

An equal volume of 50% (W/W) phenol was added to the supernatant, andstirred. The resulting suspension was centrifuged and allowed to standuntil 2 phases formed (an upper and a lower phase). The upper phase wasdialyzed exhaustively with water, then clarified by centrifugation.Next, the mixture was lyophilized. The resulting lyophilized semipureNP-9 antigen was reconstituted using 1-3 ml water and tested forantigenic activity using patient sera #1.0762 and GS in an ELISA assay.Further purification of NP-9 antigen was carried out by silica TLC usinga chloroform:methanol:0.2% CaCl₂ in water (55:45:10) solvent. NP-9antigen was then identified using patient sera #1.0762 and GS byimmunostaining TLC plates. NP-9 antigen was present in a spot or bandnear and below the position of the GM1 ganglioside. NP-9 antigen differsfrom Gal 1 ganglioside by its migration position on TLC plates, by highdegrees of binding of IgM antibodies from sera #1.0421 and 2.0328 (IgMantibody titer greater than about 1:2500 corresponds to a high degree ofbinding) and by low binding of IgM antibodies from other sera, such as#2.1343 (IgM antibody titer less than about 1:1600 corresponds to a lowdegree of binding). Patient sera #1.0762, #1.0421, #2.0328, #2.1343, andGS have been deposited with the American Type Culture Collection, 12301Parklawn Drive, Rockville, Md. 20852, U.S.A.

The purified NP-9 antigen of the present invention can be used toconstruct a synthetic NP-9 antigen, prepared by combining GM1ganglioside, cholesterol and galactocerebroside (preferably with anon-hydroxylated fatty acid moiety) in a ratio of 1:10:10 by weight.

Another embodiment of the present invention is to employ the purifiedNP-9 antigen of the present invention in a method for diagnosing aneuropathy in a patient comprising the steps of mixing serum samples ofa patient with purified NP-9 antigen; mixing serum samples of a patientwith histone H3; determining in a serum sample from a patient the titerof IgM antibodies that bind to NP-9 antigen and to histone H3; whereinhigh serum IgM antibody reactivity to NP-9 antigen (serum antibody titergreater than about 1,000), and low reactivity to histone H3 (serumantibody titer less than about 0.62 times the serum IgM antibody titerto GM1 ganglioside) correlates positively with a predominantly motorsyndrome without spasticity. This positive correlation is apparent wherethe predominantly motor syndrome without spasticity is a multifocalmotor neuropathy and is more particularly apparent where the multifocalmotor neuropathy has a clinical history of asymmetric motor weaknesswith electrophysiological evidence of motor conduction block, with orwithout axonal loss.

This positive correlation is observed where the predominantly motorsyndrome is a predominantly distal lower motor neuron disorder and, moreparticularly, where the distal lower motor syndrome has a history ofdistal asymmetric weakness which begins in a hand or a foot.

The purified NP-9 antigen of the present invention is furthercharacterized by high degrees of binding of IgM antibodies to SP neuralantigen in patient serums #1.0421 and #2.0328 (serum IgM antibody titergreater than about 1:2,500).

13.2. PATIENT TESTING

Patient sera were tested for IgM antibodies against GM1 ganglioside,NP-9 antigen, and histone H3 by Western blotting and ELISA methodology.We have identified over 70 patient sera with high titer antibodyreactivity to GM1 ganglioside (serum IgM antibody titer greater thanabout 400) and NP-9 antigen (serum IgM antibody titer greater than about1600) and with low titer IgM antibody reactivity to histone H3 (serumIgM antibody titer less than about 0.79 times the serum IgM antibodytiter to GM1 ganglioside). These patients generally have a predominantlymotor syndrome without spasticity (lower motor neuron syndromes ormultifocal motor neuropathy). We studied these patients with motorsystem disorders for patterns of antibody reactivity. Thirty-ninepatients with multifocal motor neuropathy were tested. Thirty-one ofthese had high titers of IgM antibodies to GM1 ganglioside. The positiveserums were then further tested for reactivity to histone H3 and NP-9antigen combined with MAG. Our results showed that twenty-four patientswith high titer anti-GM1 ganglioside IgM antibodies (titer greater thanabout 400) had relatively low reactivity to histone H3 (ratio of IgMantibody titers to histone H3:GM1 ganglioside less than about 0.79).Twenty-two of these serums also reacted in high titer (greater thanabout 1600) to NP-9 antigen. However, these serums did not react topurified sulfated glucuronyl paragloboside (SGPG) or to purified MAG.Reactivity could be demonstrated by Western blot and ELISA methodologyto antigens that copurified with MAG by published methodology using0.25M LIS, but not by the modification using 0.06M and then 0.25M LIS asdescribed in Section 12.1, above. The triad of reactivity of high IgMantibody titers to GM1 ganglioside and to NP-9 antigen but relativelylow reactivity to histone H3 was also found in twenty-three offorty-seven patients with distal lower motor neuron (D-LMN) syndromes.However, this triad of reactivity was only rarely present in patientswith other neurological disorders, including ALS, polyneuropathies, andautoimmune disorders. Only three of 500 disease control patients (allwith polyneuropathy) had the triad of reactivity.

Eight patients are slowly progressing weakness in their extremities withdistal onset and a diagnosis of MMN were tested for IgM antibody titersto GM-1 ganglioside, histone H3, and NP-9 antigen. They had high titersof IgM anti-GM1 ganglioside antibodies (greater than about 400) and IgManti-NP-9 antigen antibodies (greater than about 1600), and low titersof IgM anti-histone H3 antibodies (ratio of IgM antibody titers tohistone H#:GM1 ganglioside of less than about 0.79), which correlateswith MMN and distal LMN syndromes. These patients were treated by theconventional methods outlined in "A Treatable Multifocal MotorNeuropathy with Antibodies to GM1 Ganglioside," (Pestronk et al., 1988,Ann. Neurol., 24: 73-78). All had improvements in strength (Feldman etal., 1991, Ann. Neurol., 30: 397-401).

14. EXAMPLE: A MODEL OF DISEASE PRODUCTION BY INDUCING ANTISULFATIDEANTIBODIES IN EXPERIMENTAL ANIMALS

Eight guinea pigs were immunized with 0.5 mg of sulfatide mixed witheither methylated bovine serum albumin or KLH in complete Freund'sadjuvant. One month later the animals were reimmunized with a similarmixture and incomplete Freund's adjuvant. One to two weeks after thereimmunization 3 animals developed significant weakness. The illness wasterminal in two. Pathological studies show mild evidence of axonaldegeneration in peripheral nerves. Control animals immunized withmethylated BSA or KLH alone did not become ill.

15. EXAMPLE: MONOCLONAL ANTIBODIES TO NEUROPROTEIN-1

DA x Lewis hybrid Fl generation rats were immunized with NP-1 togetherwith Freund's adjuvant and hybridomas were produced using standardtechniques.

Four monoclonal antibodies, A1A1.6, A2H3.7, A2H10.1 and A5H10.1 havebeen produced. By Western blot each of these antibodies reacts with the3 bands in the NP-1 triplet (36 kD, 38 kD and 42 kD) plus a 30 kDdoublet. This suggests that the NP-1 protein bands are comprised of asingle protein with different post-translational modifications. Byimmunocytochemistry these antibodies stain fibrillary cellular processesin the central nervous system that are especially abundant in spinalgrey matter and cortex.

16. EXAMPLE MONOCLONAL ANTIBODIES TO HISTONE H3 (NEUROPROTEIN-2)

DA x Lewis hybrid Fl generation rats were immunized with histone H3(NP-2) together with Freund's adjuvant and hybridomas were producedusing standard techniques.

Four monoclonal antibodies, B3H12, B5G10, B5G12, B5H10 have beenproduced. B5H10 reacts selectively to histone H3 (NP-2) in a patternsimilar to the original W2393 test serum. In immunocytochemistry ofneural tissue the B5H10 antibody binds to cells (possibly their nuclei)in peripheral nerve and the cerebellum (especially in the granularlayer). B3H12 reacts weakly to histone H3 (NP-2) on Western blot. Athigh dilutions (1:250) it binds selectively to a 22 kD protein in thenon-myelin pellet from human brain. In neural tissue the B3H12 antibodybinds to cellular processes. In peripheral nerve axons are stronglystained. In the cerebellum processes surrounding Purkinge cells areselectively stained. B5G12 reacts equally with histone H3 (NP-2) and a22 kD protein by Western blotting methods. It also binds to a smaller10-12 kD protein. B5G10 reacts weakly with histone H3 (NP-2) on Westernblot. It binds strongly to a 46-50 kD protein in nonmyelin fractions ofhuman CNS. It binds best to an approximately 55-65 kD protein inperipheral nerve.

17. EXAMPLE: MONOCLONAL ANTIBODIES TO β-TUBULIN (NEUROPROTEIN-3)

DA x Lewis Fl generation rats were immunized with βtubulin (NP-3)together with Freund's adjuvant and hybridomas were produced usingstandard techniques.

Four monoclonal antibodies, (C1F10, C2F3, C1H3, C2H1) to β-tubulin(NP-3) have been produced. By ELISA they react strongly with NP-3 andwith tubulin extracted from the myelin pellet from human brain. Theycross-react to varying degrees with purified bovine brain tubulin.

18. EXAMPLE: MONOCLONAL ANTIBODY TO HISTONE H3

DA x Lewis hybrid Fl generation rats were immunized with histone H3together with Freund's adjuvant and hybridomas were produced usingstandard techniques. One monoclonal antibody to histone H3, 5H10, hasbeen produced. It reacts to histone H# by ELISA. 5H10 reacts to histoneH3 by Western blotting from crude preparation of histones, nervoussystem proteins, and an enriched histone preparation (f₃).

Various publications have been cited herein that are incorporated byreference in their entirety.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention and all suchmodifications are intended to be included within the scope of thefollowing claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 4                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - Met Arg Glu Ile Val Ser Ile Gln Ala Gly Gl - #n Ala Gly Asn Gln         1               5   - #                10  - #                15               - - Ile Gly Ala Lys Phe Xaa Glu Val Ile                                                      20                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Met Arg Glu Ile Val His Val Gln Ala Gly Gl - #n Cys Gly Asn Gln          1               5   - #                10  - #                15               - - Ile Gly Ala Lys Phe Trp Glu Val Ile                                                      20                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #3:                          - - Met Arg Glu Ile Val Ser Ile Gln Ala Gly Gl - #n Ala Gly Asn Gln          1               5   - #                10  - #                15               - - Ile Gly Ala Lys Phe Xaa Glu Val Ile                                                      20                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Met Arg Glu Ile Val His Val Gln Ala Gly Gl - #n Cys Gly Asn Gln          1               5   - #                10  - #                15               - - Ile Gly Ala Lys Phe Trp Glu Val Ile                                                      20                                                           __________________________________________________________________________

What is claimed is:
 1. A method for aiding in the diagnosis of apredominantly axonal peripheral neuropathy in a patient comprising:a)reacting a serum sample from the patient with sulfatide; and b)determining the titer of antisulfatide IgG antibody in the serum samplethat binds to sulfatide,wherein a titer greater than about 1:900correlates positively with a predominantly axonal peripheral neuropathy.2. The method of claim 1 in which the predominantly axonal peripheralneuropathy is predominantly sensory.
 3. The method of claim 2 in whichthe patient has a clinical history of presenting first as numbness andparesthesias or pain in the feet, and then spreading more proximately inthe legs and eventually involving the hands, then the arms.
 4. Themethod of claim 1 in which the predominantly axonal peripheralneuropathy is a pure sensory neuropathy.
 5. The method of claim 1 inwhich the predominantly axonal peripheral neuropathy is a sensory motorneuropathy.
 6. A method for aiding in the diagnosis of a predominantlyaxonal peripheral neuropathy in a patient comprising:a) reacting a serumsample from the patient with sulfatide; and b) determining the titer ofantisulfatide IgM antibody in the serum sample that binds tosulfatide,wherein a titer greater than about 1:1100 correlatespositively with a predominantly axonal peripheral neuropathy.
 7. Themethod of claim 6 in which the predominantly axonal peripheralneuropathy is predominantly sensory.
 8. The method of claim 7 in whichthe patient has a clinical history of presenting first as numbness andparesthesias or pain in the feet, and then spreading more proximately inthe legs and eventually involving the hands, then the arms.
 9. Themethod of claim 6 in which the predominantly axonal peripheralneuropathy is a pure sensory neuropathy.
 10. The method of claim 6 inwhich the predominantly axonal peripheral neuropathy is a sensory motorneuropathy.
 11. A method for aiding in the diagnosis of a predominantlyaxonal peripheral neuropathy in a patient comprising:a) reacting a serumsample from the patient with sulfatide; and b) determining the titer ofantisulfatide IgG antibody and antisulfatide IgM antibody in the serumsample that binds to sulfatide,wherein a titer of IgG antibody greaterthan about 1:900, or a titer of IgM antibody greater than about 1:1100,or both, correlates positively with a predominantly axonal peripheralneuropathy.
 12. The method of claim 11 in which the predominantly axonalperipheral neuropathy is predominantly sensory.
 13. The method of claim12 in which the patient has a clinical history of presenting first asnumbness and paresthesias or pain in the feet, and then spreading moreproximately in the legs and eventually involving the hands, then thearms.
 14. The method of claim 11 in which the predominantly axonalperipheral neuropathy is a pure sensory neuropathy.
 15. The method ofclaim 11 in which the predominantly axonal peripheral neuropathy is asensory motor neuropathy.
 16. A method for aiding in the diagnosis of apredominantly axonal peripheral neuropathy in a patient, comprising:a)determining an amount of anti-sulfatide antibody in a serum sample fromthe patient by reacting the serum sample with (i) sulfatide and (ii) adetectably labeled antibody that binds to IgG or IgM antibody; and b)comparing the amount of anti-sulfatide antibody in the serum sample fromthe patient with an amount of anti-sulfatide antibody in negativecontrol samples,wherein an amount of anti-sulfatide antibody in theserum sample from the patient that is less than 3 standard deviationsover the amount of anti-sulfatide antibody in the negative controlsamples correlates with an absence of predominantly axonal peripheralneuropathy in the patient.
 17. The method of claim 16, wherein theamount of anti-sulfatide antibody in the serum sample from the patientis determined as a titer of anti-sulfatide antibody.
 18. A method foraiding in the diagnosis of a predominantly axonal peripheral neuropathyin a patient, comprising:a) determining an amount of anti-sulfatideantibody in a serum sample from the patient by reacting the serum samplewith (i) sulfaide and (ii) a detectable labeled antibody that binds toIgG or IgM antibody; and b) comparing the amount of anti-sutlfatideantibody in the serum sample from the patient with an amount ofanti-sulfatide antibody in negative control samples,wherein an amount ofanti-sulfatide antibody in the serum sample from the patient that isgreater less than 3 standard deviations over the amount ofanti-sulfatide antibody in the negative control samples correlatespositively with a presence of predominantly axonal peripheral neuropathyin the patient.
 19. The method of claim 18, wherein the amount ofanti-sulfatide antibody in the serum sample from the patient isdetermined as a titer of anti-sulfatide antibody.
 20. A method foraiding in the diagnosis of a predominantly axonal peripheral neuropathyin a patient, comprising:a) determining an amount of anti-sulfatideantibody comprising IgG or IgM antibody in a serum sample from thepatient; and b) comparing the amount of anti-sulfatide antibody in theserum sample from the patient with an amount of anti-sulfatide antibodyin negative control samples,wherein an amount of anti-sulfatide antibodyin the serum sample from the patient that is less than 3 standarddeviations over the amount of anti-sulfatide antibody in the negativecontrol samples correlates with absence of a predominantly axonalperipheral neuropathy in the patient.
 21. The method of claim 20,wherein the amount of anti-sulfatide antibody in the serum sample fromthe patient is determined as a titer of anti-sulfatide antibody.
 22. Amethod for aiding in the diagnosis of a predominantly axonal peripheralneuropathy in a patient, comprising:(a) determining an amount ofanti-sulfatide antibody comprising IgG or IgM antibody in a serum samplefrom the patient; and (b) comparing the amount of anti-sulfatideantibody in the serum sample from the patient with an amount ofanti-sulfatide antibody in negative control samples,wherein an amount ofanti-sulfatide antibody in the serum sample from the patient that isgreater than 3 standard deviations over the amount of anti-sulfatideantibody in the negative control samples correlates positively with apredominantly axonal peripheral neuropathy in the patient.
 23. Themethod of claim 22, wherein the amount of anti-sulfatide antibody in theserum sample from the patient is determined as a titer of anti-sulfatideantibody.
 24. The method of claim 16, wherein the sulfatide isimmobilized on a solid substrate.
 25. The method of claim 18, whereinthe sulfatide is immobilized on a solid substrate.
 26. A method foraiding in the diagnosis of a predominantly axonal peripheral neuropathyin a patient, comprising:a) determining a titer of anti-sulfatideantibody comprising IgG antibody in a serum sample from the patient byreacting the serum sample with (i) sulfatide and (ii) a detectablylabeled antibody that binds to IgG antibody; and b) comparing the titerof anti-sulfatide antibody in the serum sample from the patient with atiter of anti-sulfatide antibody in a negative control sample,wherein atiter of anti-sulfatide IgG antibody in the serum sample from thepatient that is less than about 1:900 correlates with an absence ofpredominantly axonal peripheral neuropathy in the patient.
 27. A methodfor aiding in the diagnosis of a predominantly axonal peripheralneuropathy in a patient, comprising:a) determining a titer ofanti-sulfatide antibody comprising IgM antibody in a serum sample fromthe patient by reacting the serum sample with (i) sulfatide and (ii) adetectably labeled antibody that binds to IgM antibody; and b) comparingthe titer of anti-sulfatide antibody in the serum sample from thepatient with a titer of anti-sulfatide antibody in a negative controlsample,wherein a titer of anti-sulfatide IgM antibody in the serumsample from the patient that is less about 1:1100 correlates with anabsence of predominantly axonal peripheral neuropathy in the patient.28. A method for aiding in the diagnosis of a predominantly axonalperipheral neuropathy in a patient, comprising:a) determining a titer ofanti-sulfatide antibody comprising IgG antibody in a serum sample fromthe patient by reacting The serum sample with (i) sulfatide and (ii) adelectably labeled antibody that binds to IgG antibody; and b) comparingthe titer of anti-sulfatide antibody in the serum sample from thepatient with a titer of anti-sulfatide antibody in a negative controlsample,wherein a titer of anti-sulfatide IgG antibody in the serumsample from the patient that is greater than about 1:900 ofanti-sulfatide IgG antibody in the negative control sample correlatespositively with a presence of predominantly axonal peripheral neuropathyin the patient.
 29. A method for aiding in the diagnosis of apredominantly axonal peripheral neuropathy in a patient, comprising:a)determining a titer of anti-sulfatide antibody comprising IgM antibodyin a serum sample from the patient by reacting the serum sample with (i)sulfatide and (ii) a detectably labeled antibody that binds to IgMantibody; and b) comparing the titer of anti-sulfatide antibody in theserum sample from the patient with a titer of anti-sulfatide antibody ina negative control sample,wherein a titer of anti-sulfatide IgM antibodyin the serum sample from the patient that is greater than about 1:1100of anti-sulfatide IgM antibody in the negative control sample correlatespositively with a presence of predominantly axonal peripheral neuropathyin the patient.
 30. A method for aiding in the diagnosis of apredominantly axonal peripheral neuropathy in a patient, comprising:a)determining a titer of anti-sulfatide antibody comprising IgG antibodyin a serum sample from the patient; and b) comparing the titer ofanti-sulfatide antibody in the serum sample from the patient with atiter of anti-sulfatide antibody in a negative control sample,wherein atiter of anti-sulfatide IgM antibody in the serum sample from thepatient that is less than about 1:900 correlates with an absence ofpredominantly axonal peripheral neuropathy in the patient.
 31. A methodfor aiding in the diagnosis of a predominantly axonal peripheralneuropathy in a patient, comprising:a) determining a titer ofanti-sulfatide antibody comprising IgM antibody in a serum sample fromthe patient; and b) comparing the titer of anti-sulfatide antibody inthe scram sample from the patient with a titer of anti-sulfatideantibody in a negative control sample,wherein a titer of anti-sulfatideIgM antibody in the serum sample from the patient that is less thanabout 1:1100 correlates with an absence of predominantly axonalperipheral neuropathy in the patient.
 32. A method for aiding in thediagnosis of a predominantly axonal peripheral neuropathy in a patient,comprising:a) determining a titer of anti-sulfatide antibody comprisingIgG antibody in a serum sample from the patient; and b) comparing thetiter of anti-sulfatide antibody in the serum sample from the patientwith a titer of anti-sulfatide antibody in a negative controlsample,wherein a titer of anti-sulfatide IgM antibody in the serumsample from the patient that is greater than about 1:900 ofanti-sulfatide IgG antibody in the negative control sample correlatespositively with a presence of predominantly axonal peripheral neuropathyin the patient.
 33. A method for aiding in the diagnosis of apredominantly axonal peripheral neuropathy in a patient, comprising:a)determining a titer of anti-sulfatide antibody comprising IgM antibodyin a serum sample from the patient; and b) comparing the titer ofanti-sulfatide antibody in the serum sample from the patient with atiter of anti-sulfatide antibody in a negative control sample,wherein atiter of anti-sulfatide IgM antibody in the serum sample from thepatient that is greater than about 1:1100 of anti-sulfatide IgM antibodyin the negative control sample correlates positively with a presence ofpredominantly axonal peripheral neuropathy in the patient.